PCR assays for the detection of members of the ...

Journal of Virological Methods 165 (2010) 97–104

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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae Milton Untiveros 1 , Zoila Perez-Egusquiza ∗ , Gerard Clover Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, P.O. Box 2095, Auckland 1140, New Zealand

a b s t r a c t Article history: Received 14 September 2009 Received in revised form 12 January 2010 Accepted 20 January 2010 Available online 1 February 2010 Keywords: Bromoviridae Ilarviruses Universal primers RT-PCR

A PCR assay was developed for the universal detection of ilarviruses using primers designed to the RNAdependent RNA polymerase gene in RNA2. The assay detected 32 isolates of 15 definite and 2 tentative ilarvirus species using a one-step RT-PCR. The assay was more specific, and at least as sensitive as a commercial assay, and allowed direct sequencing of amplicons. No cross-reaction was observed with neither healthy plants of 15 host species nor from isolates in other genera of the Bromoviridae. A further PCR assay targeting the helicase motif of RNA1 was able to detect all species tested within the family Bromoviridae, including members of the Alfamovirus, Anulavirus, Bromovirus, Cucumovirus and Ilarvirus. The assays provide a sensitive and cost-effective way for detecting and characterising members of the Bromoviridae and can be used for quarantine and certification programmes. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The family Bromoviridae is one of the most important families of plant viruses and its members infect a wide range of hosts including herbaceous plants, shrubs and trees. Taxonomically the family is divided into six genera: Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus and Oleavirus (Fauquet et al., 2005; Carstens and Ball, 2009). With more than 16 species divided into six subgroups, the genus Ilarvirus is by far the largest genus of the family. As with all genera in the Bromoviridae, the ilarviruses have a tripartite positive-strand RNA genome encoding four or five proteins. RNA1 is monocistronic and encodes the viral replicase which has methyltransferase and helicase signature motifs. RNA2 can be mono or bicistronic (Xin et al., 1998; Shiel and Berger, 2000) and encodes the 2a protein, an RNA-dependent RNA polymerase (RdRp). In some species of subgroups 1 and 2, a second gene is present (Xin et al., 1998). This gene, 2b is expressed in vivo through a subgenomic mRNA and, similar to the 2b protein of cucumoviruses, is thought to be involved in suppression of RNA silencing (Brigneti et al., 1998) and in cell-to-cell movement (Xin et al., 1998). Neither alfamoviruses nor bromoviruses are reported to encode this ORF. RNA3 encodes the movement protein (MP) and coat protein (CP). While the MP is expressed directly from this RNA, CP synthesis occurs via a monocistronic and subgenomic RNA4. In addition to forming the viral capsid, this protein is required for virus move-

∗ Corresponding author. Tel.: +64 9 9095709; fax: +64 9 9095739. E-mail address: [email protected] (Z. Perez-Egusquiza). 1 Present address: International Potato Center, CIP P.O. Box, 1558-Lima 12, Peru. 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.01.011

ment and genome activation (Jaspars, 1999; Neeleman et al., 2004). The requirement of the CP for genome activation is shared by all ilarviruses and Alfalfa mosaic virus (AMV) but is not found in other member of the family (Bol, 1999). Most ilarviruses have a wide host range and infect predominantly woody perennials (Vaskova et al., 2000; Zimmerman and Scott, 2001; Fauquet et al., 2005). They cause diseases of economic importance in Citrus, Humulus, Malus, Prunus, Rosa and Rubus spp., affecting plant growth, and fruit yield and maturity (Uyemoto and Scott, 1992; Saade et al., 2000; Tzanetakis et al., 2007). The vector(s) and mechanism of transmission of most ilarviruses are still unknown, although at least seven species are pollen- and/or seedtransmitted (Card et al., 2007). Insects may be involved in mediating the movement of virus-infected pollen from the flowers of diseased plants to those of healthy plants, e.g. thrips with Tobacco streak virus (TSV) (Kaiser et al., 1982), Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV) (Greber et al., 1992); and honeybees with Blueberry shock virus (BlShV) (Bristow and Martin, 1999). This lack of information is probably due in part to the difficulty in handling and manipulating ilarviruses, in particular they are difficult to isolate from their woody hosts and their mechanical transmission is not easy. The most effective measures to prevent the damage caused by ilarvirus infection are by quarantine and/or using virus-free planting material. This is especially important since at least some ilarviruses are transmitted by pollen and therefore they can spread rapidly over long distances, and many of the host species are perennials. Moreover, ilarviruses occur often in mixed infections and are present in low titre in their hosts (Uyemoto and Scott, 1992). Consequently, considerable effort has been made to develop proce-

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M. Untiveros et al. / Journal of Virological Methods 165 (2010) 97–104

dures that allow sensitive and simultaneous detection of different ilarviruses. Techniques such as non-isotropic molecular hybridisation, one- and two-step RT-PCR, multiplex and generic ramped annealing RT-PCR have been evaluated (Saade et al., 2000; Scott et al., 2003; Herranz et al., 2005; Sanchez-Navarro et al., 2005; Maliogka et al., 2007). Although these methods are sensitive and reliable, they do not detect all species in the genus and/or their complexity means that they are not suited for routine applications. Currently the detection method used most commonly is a RT-PCR method using commercial ilarvirus-group primers (Agdia Inc., Elkhart, IN, USA), e.g. Maroon-Lango et al. (2006). However, the PCR conditions are long and cloning of PCR amplicon is required to identify the virus species by sequencing (direct sequencing is not possible since forward and reverse primers are mixed in the Agdia kit). This paper describes the results of experiments to develop a reliable, sensitive, and specific method to detect ilarviruses cheaply and quickly. Conserved sequences were identified and used to design primer pairs that amplified specific fragments from 17 ilarvirus species under standard one-step PCR reaction conditions. In addition, a primer pair was designed to detect all members of the Bromoviridae family. The advantages of this approach compared to existing tests for ilarviruses and Bromoviridae are discussed. 2. Materials and methods 2.1. Virus isolates The virus isolates used during study were obtained either from commercial sources or researchers as fresh or freeze-dried leaves, nucleic acids or viral purified preparations (Table 1). 2.2. Design of oligonucleotides Ilarvirus-specific primers were designed based on genome sequences obtained from the GenBank database (http://www.ncbi.nlm.nih.gov) in February 2009 (Table 1). Sequences of RNA1, RNA2 and RNA3 of the selected isolates were aligned using the MEGA 4.0 software (Kumar et al., 2004). Conserved regions were identified visually and primers for these regions were designed. The thermodynamic and structural properties of these primers were analysed using Oligo Explorer 1.1.0 (http://molbiol-tools.ca/molecular biology freeware.htm). Additional representative genome sequences of viruses in other genera of the Bromoviridae family (Table 1) were aligned to verify the specificity of the primers in silico. 2.3. RNA extraction and cDNA synthesis Total RNA was extracted from infected samples using an RNeasy® Plant Mini Kit (Qiagen, Doncaster, Australia) following the manufacturer’s protocol and stored at −80 ◦ C. Reverse transcription reactions were done using SuperScript III Reverse Transcriptase (Invitrogen) with random hexamer primers following the manufacturer’s instructions. In the first step of cDNA synthesis, 4 ␮l total RNA, 0.1 ␮l (40 U/␮l) RNasin® Plus RNase Inhibitor, 4 ␮l 5× first strand buffer, 0.5 ␮l (0.5 ng/␮l) random hexamer primers, 2 ␮l (10 ␮g/␮l) bovine serum albumin (BSA) (Sigma–Aldrich, Auckland, New Zealand) and 5.4 ␮l nuclease-free water were mixed and denatured at 70 ◦ C for 10 min and then kept at room temperature for 15 min. To this mixture, 0.5 ␮l (200 U/␮l) SuperScript III Reverse Transcriptase, 0.1 ␮l (40 U/␮l) RNasin® Plus RNase Inhibitor, 2 ␮l 0.1 M DTT, 1.0 ␮l 10 mM dNTPs and 0.4 ␮l nuclease-free water were added before incubating at 42 ◦ C for 1 h. The synthesized cDNA was stored at −20 ◦ C.

2.4. Primer testing and optimization of the RT-PCR conditions Seven combinations of novel primers were investigated in one and two-step PCR reactions. The primer pairs were named Ilar1F2/Ilar1R3 and Ilar1F5/Ilar1R7 (targeting RNA1), and Ilar2F4/Ilar2R9, Ilar2F5/Ilar2R9, Ilar2F5/Ilar2R8, Ilar2F6/Ilar2R8 and Ilar2F6/Ilar2R9 (targeting RNA2). When designing the assays, the primer melting temperatures, formation of heterodimer complexes, and size of the expected amplicon were considered. Oneand two-step RT-PCR assays were done using either the VersoTM 1-Step RT-PCR Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA) or GoTaq® DNA polymerase (Promega, Madison, WI, USA), respectively, following the manufacturer’s instructions. The assays were done in 20 ␮l reaction volumes containing either 1 ␮l total RNA (one-step RT-PCR) or 1 ␮l cDNA (two-step RT-PCR). BSA (RIA grade, A7888-50G; Sigma–Aldrich, St. Louis, MO, USA) was added to the one-step RT-PCR reactions at a final concentration of 0.5 ␮g/␮l. The following conditions were optimized for two-step RT-PCR: (1) primer concentrations (final concentrations at 0.5, 1 or 3 ␮M); (2) annealing temperature (40–55 ◦ C) and time (30 s to 2 min); and (3) extension time (from 30 s to 2 min). Gradient PCR was performed using a Palm-CyclerTM machine (Corbett Research, Sydney, Australia) to establish the optimal annealing and extension temperatures. cDNA of TSV was used initially to optimize PCR conditions for each primer set. One-step RT-PCR was done using an RT step of 50 ◦ C for 20 min followed by the optimal PCR conditions. 2.5. Specificity and sensitivity of the developed detection assays The specificity of the novel primer sets was compared to those of Maliogka et al. (2007) and commercial Agdia primers using 32 isolates from 17 ilarvirus species and additional isolates of Alfalfa mosaic virus (AMV), Brome mosaic virus (BMV), Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV) (Table 1). Amplicons of the expected size were sequenced directly in both directions to verify their identity and the sequences were deposited in the GenBank database. In addition, total RNA from 15 healthy species (Chenopodium quinoa, Citrus sinensis × Poncirus trifoliata, Fragaria × ananassa, Humulus lupulus, Malus × domestica, Nicotiana tabacum, Prunus armeniaca, P. domestica, P. persicae, Rosa sp., Ribes nigrum, Rubus fructicosus, R. idaeus, Solanum lycopersicum, Vaccinium corymbosum), which are susceptible to ilarvirus infection, were tested using the same RT-PCR conditions to investigate whether there was any non-specific reaction. To determine the sensitivity of the novel primer sets in onestep PCR, a series of 10-fold dilutions (ranging from 1 to 10−4 ) of viral RNA from three ilarviruses (Parietaria mottle virus [PMoV], Spinach latent virus [SpLV] and American plum line pattern virus [APLPV]) belonging to different subgroups were tested using the novel assay and the commercial Agdia assay following the manufacturer’s instructions. Dilutions were made using RNA extracted from healthy plants of corresponding species. 3. Results 3.1. Sequence analysis and primer design Fifty genomic sequences targeting the three RNAs of 16 ilarviruses were analysed. Sequence alignment enabled the identification of four and five conserved regions suitable for primer design in RNA1 and RNA2, respectively. Within RNA1, primers were designed to conserved regions including the methyltransferase and helicase signature motifs. The primers targeting RNA2 were located


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Table 1 List of isolates and genomic sequences used in this study. Genus

Species (acronym)

Isolate ID/Supplier

Host

Ilarvirus subgroup 1

Tobacco streak virus (TSV)

– PV-0738/DSMZ, Germany PV-0615/DSMZ, Germany PV-0612/DSMZ, Germany PV-0309/DSMZ, Germany – University of California, USA PV-569/ATCC, USA PV-0715/DSMZ, Germany PV-0400/DSMZ, Germany – University of Arkansas, USA

– Chenopodium quinoa Chenopodium quinoa Chenopodium quinoa Chenopodium quinoa – Fragaria sp. Fragaria sp. – Solanum lycopersicum Parietaria officinalis – Rosa sp.

– PV-0953/DSMZ, Germany LPC 71000/Agdia, USA – IVIA, Spain FM-23/IVIA, Spain – MAF, New Zealand – PV-80/ATCC, USA – Clemson University, USA – Clemson University, USA

– Asparagus officinalis Unknown – Citrus sp. Citrus sp. – Chenopodium quinoa – Chenopodium quinoa – Chenopodium quinoa – Unknown

– H8/MAF, New Zealand G9/MAF, New Zealand PSA 30500/Agdia, USA – PSA 30700/Agdia, USA ApM-LPC/Sediag, France PV-0742/DSMZ, Germany – Clemson University, USA USDA, USA LPC 19200/Agdia, USA

– Chenopodium quinoa Unknown Unknown – Unknown Unknown Rubus sp. – Humulus lupulus Vaccinium stamineum Unknown

Strawberry necrotic shock virus (SNSV)

Parietaria mottle virus (PMoV)

Blackberry chlorotic ringspot virus (BCRV) Ilarvirus subgroup 2

Asparagus virus 2 (AV-2)

Citrus variegation virus (CVV)

Spinach latent virus (SpLV) Tulare apple mosaic virus (TAMV) Elm mottle virus (EMoV) Citrus leaf rugose virus (CiLRV) Ilarvirus subgroup 3

Prunus necrotic ringspot virus (PNRSV)

Apple mosaic virus (ApMV)

Humulus japonicus latent virus (HJLV) Blueberry shock virus (BlShV)

GenBank accession number RNA1

RNA2

RNA3

U80934

U75538

X00435

DQ318818

AY743591

AY363228

AY496068

AY496069

NC 005854

DQ091193

DQ091194

DQ091195

EU919666

EU919667

X86352

EF584664

EF584665

U17389

U93192

U93193

U93194

AF226160

AF226161

AF226162

U57047

U34050

U85399

U23715

U17726

U17390

AF278534

AF278535

U57046

AF174584

AF174585

U15608

AY500236

AY500237

AY500238


Prune dwarf virus (PDV)

– LPC 98700/Agdia, USA PV-0804/DSMZ, Germany PV-0523/DSMZ, Germany

– Unknown Prunus armeniaca Prunus armeniaca

U57648

AF277662

L28145


American plum line pattern virus (APLPV)

– CFIA, Canada APLP-LPC/Sediag, France

– Prunus domestica Unknown

AF235033

AF235165

AF235166


Fragaria chiloensis latent virus (FClLV)

– University of Arkansas, USA

– Unknown

AY682102

AY707771

AY707772

Alfamovirus

Alfalfa mosaic virus (AMV)

– MAF, New Zealand

– Viburnum sp.

L00163

L00161

L00162

Anulavirus

Pelargonium zonate spot virus (PZSV)

– LPC 90100/Agdia, USA

– Unknown

NC 003649

NC 003650

NC 003651

Bromovirus

Brome mosaic virus (BMV)

– LPC 29300/Agdia, USA – – –

– Unknown – – –

X02380

X01678

V00099

M65138 M65139 AB080598

M64713 M28817 AB080599

M60291 M28818 AB080600

– – PSA 44501/Agdia, USA – – LPC44600/Agdia, USA – PSA 37000/Agdia, USA

– – Unknown – – Unknown – Unknown

D00356 X02733

D00355 X00985

D10538 M21463

U15728 D11126

U15729 D11127

U15730 D00668

D10663

D10044

D01015

–

–

X94346

–

X77115

Broad bean mottle virus (BBMV) Cowpea chlorotic mottle virus (CCMV) Spring beauty latent virus (SBLV) Cucumovirus

Cucumber mosaic virus (CMV), Fry strain CMV, Q Strain Peanut stunt virus (PSV), ER strain PSV, J strain Tomato aspermy virus (TAV)

Oleavirus

Olive latent virus 2 (OLV-2)

100


Table 2 PCR primers designed during the current study (Tm, melting temperature). The position numbers refer to bases in RNA1 and RNA2 of Tobacco streak virus (GenBank accession numbers U80934 and U75538, respectively). Target

Name

Sense

Sequence

Position

Length

Tm

Degeneracy

RNA1

Ilar1F2 Ilar1F3 Ilar1R3 Ilar1F5 Ilar1R5 Ilar1R7

Sense Sense Antisense Sense Antisense Antisense

AAYGTBCAYWSNTGYTGYCC TTYSAYTTYRWBGATGCHCC GGDGCATCVWYRAARTSRAA GCNGGWTGYGGDAARWCNAC GTNGWYTTHCCRCAWCCNGC AMDGGWAYYTGYTYNGTRTCACC

452–471 809–828 809–828 2459–2478 2459–2478 2750–2772

20 19 19 20 20 21

52.2 52 52 54.8 54.8 53.6

576 768 768 768 768 3072

RNA2

Ilar2F4 Ilar2F5 Ilar2F6 Ilar2R6 Ilar2R8 Ilar2R9

Sense Sense Sense Antisense Antisense Antisense

CBATHACHTATCAYRADAARGG TCRAYRTTYGAYAARTCNCA GGWGATGCNDRYACNTAYYT ARRTANGTRYHNGCATCWCC CCAATNARRSWRTCRTCDCC GGTTGRTTRTGHGGRAAYTT

1258–1279 1440–1459 1614–1633 1614–1633 1716–1735 1800–1819

22 20 20 20 20 20

46.1 47.3 46.4 46.4 49.6 48.8

648 512 1536 1536 768 48

in the RdRp gene. No conserved region suitable for primer design could be identified in RNA3. The nucleotide sequences and features of the primers are summarized in Table 2. Analysis of a further 30 sequences of species in other genera in the Bromoviridae revealed that some regions in RNA1 but not RNA2, were conserved throughout the family. Consequently the primers designed to detect RNA1 of ilarviruses may have a broader specificity (data not shown). 3.2. Primer testing and optimization of the RT-PCR conditions Only three primer pairs produced amplicons from at least one member of each ilarvirus subgroup under the optimal conditions described in Table 3. The primer pairs Ilar2F5/Ilar2R9 and Ilar1F5/Ilar1R7 amplified PCR products of the expected size of ∼380 and 300 bp, respectively from 17 ilarviruses (Fig. 1a and c; data for CiLRV not shown). The third pair, Ilar2F4/Ilar2R9, yielded a 560 bp amplicon with most samples but only weak amplification was observed with one isolate of Apple mosaic virus (ApMV) (Fig. 1b). The three primer pairs were able to amplify those ilarviruses using either one or two-step RT-PCR. The optimized PCR conditions are described in Table 3. 3.3. Specificity and sensitivity of the developed RT-PCR assays Amplicons of the expected size were obtained from all samples of 32 isolates of 17 species of ilarviruses using the Ilar2F5/Ilar2R9 primers (Table 4). Sequences of the ∼380 bp amplicons were concordant with published RdRp sequences of each ilarvirus (Table 4),

except one isolate of TSV was reported to have a closer identity to Strawberry necrotic shock virus (SNSV). No sequence has been published for BlShV but the closest identity was 77% to PNRSV. No non-specific products were amplified using primer pair Ilar2F5/Ilar2R9 neither from healthy plants of any of the 15 host species tested nor from isolates in other genera of the Bromoviridae (data not shown). Comparison of the sensitivity of the Ilar2F5/Ilar2R9 primer pair and the Agdia primers using a dilution series of RNA extractions of PMoV (Subgroup 1), SpLV (Subgroup 2) and APLPV (Subgroup 5), resulted detection of 10−4 dilution of each of the three viruses (Fig. 2). The primer pair Ilar1F5/Ilar1R7 detected all ilarvirus isolates in one-step PCR assays; however, these primers were not ilarvirusspecific and detected viruses from other genera in the Bromoviridae (Fig. 1c, Table 4). No specific bands were observed when other ss RNA (+) viruses were tested using these primers (data not shown except Pea enation mosaic virus [Fig. 1c]). However, nonspecific products were amplified occasionally from healthy plants but these were generally of a different size than those from infected plants (data not shown). The primer pair Ilar2F4/Ilar2R9 detected at least one isolate of each ilarvirus species (Fig. 1c) but failed to amplify some isolates of APLPV, ApMV and PDV (data not shown). The primers of Maliogka et al. (2007) detected all 17 ilarvirus species except APLPV (data not shown). The Agdia primers detected all 17 ilarvirus species but also cross-reacted with BMV (data not shown).

Table 3 Primer sets, target regions and PCR conditions. Primer pair

Ilar2F5/Ilar2R9

Ilar1F5/Ilar1R7

Ilar2F4/Ilar2R9

Final primer concentration

1/0.5 ␮M

1/1 ␮M

1/0.5 ␮M

Location

RdRp gene, RNA2

Methyltransferase motif, RNA1

RdRp gene, RNA2

Size

PCR cycling conditions

∼380 bp

94 ◦ C, 5 min 94 ◦ C, 30 s 44 ◦ C, 1 min 72 ◦ C, 1 min 72 ◦ C, 10 min 94 ◦ C, 5 min 94 ◦ C, 30 s 47.7 ◦ C, 30 s 72 ◦ C, 30 s 72 ◦ C, 10 min

∼300 bp

∼560 bp

40 cycles

94 ◦ C, 5 min 94 ◦ C, 30 s 48.7 ◦ C, 1 min 72 ◦ C, 1 min 72 ◦ C, 5 min

40 cycles

40 cycles


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Fig. 1. Comparison of different primer combinations for the detection of ilarviruses: (a) primers Ilar2F5/Ilar2R9 (380 bp), (b) primers Ilar2R4/Ilar2R9 (560 bp) and (c) Ilar1F5/Ilar1R7 (∼300 bp) primers. In (a and b) lanes 1–18 represent Tobacco streak virus, Citrus variegation virus, Apple mosaic virus, American plum line pattern virus, Prunus necrotic ringspot virus, Prune dwarf virus, Humulus japonicus latent virus, Tulare apple mosaic virus, Strawberry necrotic shock virus, Spinach latent virus, Asparagus virus 2, Parietaria mottle virus, Elm mottle virus, Blackberry chlorotic ringspot virus, Fragaria chiloensis latent virus, Blueberry shock virus, negative water control and Alfalfa mosaic virus; additionally in (a) lanes 19–21 represent Cucumber mosaic virus, Brome mosaic virus and Peanut stunt virus. In (c) lanes 1–14 represent Tobacco streak virus, Citrus variegation virus, Apple mosaic virus, Prune dwarf virus, American plum line pattern virus, Fragaria chiloensis latent virus, Alfalfa mosaic virus, Cucumber mosaic virus, Brome mosaic virus, Peanut stunt virus, Pelargonium zonate spot virus, Tomato aspermy virus, Pea enation mosaic virus and negative water control. M: 100 bp DNA ladder (Invitrogen).

4. Discussion The use of universal primers to detect related viral species in one PCR assay has been demonstrated to be an effective strategy for rapid and accurate virus detection. This approach is used routinely for the detection of genera such as closteroviruses (Saldarelli et al., 1998), foveaviruses and vitiviruses (Dovas and Katis, 2003), potexviruses (van der Vlugt and Berendsen, 2002) and potyviruses (Mackenzie et al., 1998). Previous researchers have developed PCR methods for detection of ilarviruses. The use of multiplex RT-PCR with primers targeting RNA3 has been reported for detection of four species from different subgroups affecting stonefruit (Saade et al., 2000; Sanchez-Navarro et al., 2005). Scott et al. (2003) developed primers targeting RNA1 and RNA2 to detect members of subgroup 2. Maliogka et al. (2007) designed degenerate nested primers in RNA2, and using ramped annealing RT-PCR detected eight species from different subgroups. In contrast to previous reports, the primer sets in the current study detected multiple

isolates of 17 ilarviruses using one-step RT-PCR. The primer set targeting RNA1 was also able to detect species from other genera in the family Bromoviridae. The Ilar2F5/Ilar2R9 primer pair was designed to detect motifs A (S[K/T]FDKS) and D (KFP[H/Y]NQ) of the polymerase core palm structure in RNA2 (O’Reilly and Kao, 1998), and was the most specific for the detection of ilarviruses. These primers detected all 32 isolates of the 17 ilarvirus species tested (Table 1). These species included all definite members of the genus except Lilac ring mottle virus (no isolate of this virus is available) and two tentative ilarviruses, Blackberry chlorotic ringspot virus and SNSV. There was no non-specific reaction with healthy tissue when this was assessed using healthy plants from 15 host species. In contrast, the primers of Maliogka et al. (2007) did not detect APLPV, while the Agdia primers cross-reacted with BMV. Unlike the Agdia and Maliogka et al. (2007) assays the new primer pair could be used in one-step RT-PCR, thereby reducing the risk of contamination. The sensitivity of the Ilar2F5/Ilar2R9 primer pair was comparable to the

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Table 4 Comparison of the sequence of the PCR products amplified by the newly designed primers with those in GenBank. Species (acronym)

Ilarvirus Tobacco streak virus (TSV)

Strawberry necrotic shock virus (SNSV) Parietaria mottle virus (PMoV) Blackberry chlorotic ringspot virus (BCRV) Asparagus virus 2 (AV-2) Citrus variegation virus (CVV) Spinach latent virus (SpLV) Tulare apple mosaic virus (TAMV) Elm mottle virus (EMoV) Citrus leaf rugose virus (CiLRV) Prunus necrotic ringspot virus (PNRSV)

Apple mosaic virus (ApMV)

Humulus japonicus latent virus (HJLV) Blueberry shock virus (BlShV) Prune dwarf virus (PDV)

American plum line pattern virus (APLPV) Fragaria chiloensis latent virus (FClLV) Bromoviridae Alfalfa mosaic virus (AMV) Brome mosaic virus (BMV) Pelargonium zonate spot virus (PZSV) Cucumber mosaic virus (CMV) Peanut stunt virus (PSV) Tomato aspermy virus (TAV)

Isolate ID/supplier

Product size (bp)

GenBank accession no.

Virus, accession number, nucleotide sequence identity

PV-0738/DSMZ, Germany PV-0615/DSMZ, Germany PV-0612/DSMZ, Germany PV-0309/DSMZ, Germany University of California, USA PV-569/ATCC, USA PV-0715/DSMZ, Germany PV-0400/DSMZ, Germany University of Arkansas, USA PV-0953/DSMZ, Germany LPC 71000/Agdia, USA IVIA, Spain FM-23/IVIA, Spain MAF, New Zealand PV-80/ATCC, USA Clemson University, USA Clemson University, USA P03H8/MAF, New Zealand P03G9/MAF, New Zealand PSA 30500/Agdia, USA PSA 30700/Agdia, USA ApM-LPC/Sediag, France PV-0742/DSMZ, Germany Clemson University, USA USDA, USA LPC 19200/Agdia, USA LPC 98700/Agdia, USA PV-0804/DSMZ, Germany PV-0523/DSMZ, Germany CFIA, Canada APLP-LPC/Sediag, France University of Arkansas, USA

383 383 382 382 375 379 384 384 382 384 380 384 383 383 384 384 388 384 383 384 384 383 384 386 382 380 380 379 380 380 382 380

GQ865684 GQ865647 GQ865648 GQ865649 GQ865650 GQ865651 GQ865652 GQ865653 GQ865654 GQ865655 GQ865656 GQ865657 GQ865658 GQ865659 GQ865660 GQ865661 GQ865662 GQ865663 GQ865664 GQ865665 GQ865666 GQ865667 GQ865668 GQ865669 GQ865670 GQ865671 GQ865672 GQ865673 GQ865674 GQ865675 GQ865676 GQ865677

TSV, U75538.1, 98% TSV, U75538.1, 87% TSV, AM412234, 99% SNSV, AY743591, 79% SNSV, AY743591, 100% SNSV, AY743591, 98% PMoV, AY496069, 96% PMoV, AY496069, 90% BCRV, DQ091194, 82% AV-2, EU919667, 97% AV-2, EU919667, 97% CVV, EF584665, 95% CVV, EF584665, 95% SpLV, U93193, 98% TAMV, AF226161, 98% EMoV, U34050, 98% CiLRV, U17726.1, 98% PNRSV, AF278535, 97% PNRSV, AF278535, 90% PNRSV, AF278535, 97% ApMV, AM412229, 89% ApMV, AM412229, 94% ApMV, AM412229, 96% HJLV, AY500237, 98% PNRSV, AF278535, 77% PNRSV, AF278535.1, 76% ApMV, AF277662, 93% ApMV, AF277662, 93% ApMV, AF277662, 91% APLPV, AF235165, 98% APLPV, AF235165, 98% FClLV, AY707771, 98%

MAF, New Zealand LPC 29300/Agdia,USA LPC 90100/Agdia, USA PSA 44501/Agdia, USA LPC 44600/Agdia, USA PSA 37000/Agdia, USA

309 296 290 291 292 298

GQ865678 GQ865679 GQ865680 GQ865681 GQ865682 GQ865683

AMV, L00163.1, 92% BMV, DQ530423.1, 97% PZSV, AJ272327.1, 98% CMV, D00356.1, 96% PSV, U15728.1, 95% TAV, D10044.1 96%

Fig. 2. Comparison of the detection limits between newly designed primers Ilar2F5/Ilar2R9 (top) and ilarvirus-specific primers from Agdia (bottom). Lanes 1–5, 6–10, 11–15, represented 10 times serial dilutions of Parietaria mottle virus, Spinach latent virus and American plum line pattern virus, respectively. Lane M: 100 bp DNA ladder (Invitrogen).


Agdia primers (Fig. 2). In contrast to the commercial primers, the Ilar2F5/Ilar2R9 primers can be used for direct sequencing without the need to first clone the amplicon, thereby allowing quicker and cheaper identification. In addition, the duration of the PCR reaction is shorter than the Agdia assay (approximately 2 h less) and the primers are considerably cheaper. Therefore the new primers are considered more suitable for routine diagnosis than other methods available currently. The Ilar2F5/Ilar2R9 primer pair only amplified ilarviruses and not species in other genera of the Bromoviridae. With the exception of ilarviruses and Alfalfa mosaic virus (AMV), all Bromoviridae have an insertion of a codon for proline between the 11 and 12th bases of the reverse primer Ilar2R9. AMV differs from ilarviruses in the key 4th and 5th positions of the 3 -end of the forward primer Ilar2F5, and this probably explains why the primer pair does not detect the virus. The insertion of the proline codon in the Ilar2R9 primer may also explain why the Ilar2F4/Ilar2R9 primer pair was ilarvirus-specific. However, this pair did not detect some isolates of ApMV, PDV and APLPV. The Ilar2F4 primer was designed to a non-specific motif of the polymerase core palm structure; RNA2 of ApMV isolates is highly variable (Saade et al., 2000; Lee et al., 2002). Subgroups 4 and 5 (to which APLPV and PDV belong) are ˜ considered the most divergent subgroups in the genus (Codoner and Elena, 2006); this variability may explain the lack of amplification. The sequences of the products amplified using the Ilar2F5/Ilar2R9 primer pair correlated with the expected viral species (Table 4) with two exceptions. The closest identity for the PV-0309 isolate of TSV was 79% with SNSV (GenBank accession no. AY743591). However, for many years SNSV was considered an isolate of TSV and only recently has it been classified as a distinct species in subgroup 1 (Tzanetakis et al., 2004). No sequence for BlShV is available on GenBank therefore no comparison could be made to isolates sequenced previously. The two isolates of this species shared 76–77% identity with PNRSV. BlShV has been reported to be related serologically to PNRSV (MacDonald et al., 1991) and both species are classified as members of subgroup 3. Universal assays which detect all species in a plant virus family are unusual. The only assays used routinely are those for the Potyviridae family, e.g. Marie-Jeanne et al. (2000). In this study, the Ilar1F5/Ilar1R7 primer set detected a wide range of species in the Bromoviridae family including ilarviruses, AMV in the genus Alfamovirus, PZSV in the genus Anulavirus, BMV in the genus Bromovirus, and CMV, Peanut stunt virus and Tomato aspermy virus in the genus Cucumovirus. This primer pair is designed to detect RNA1. The forward primer Ilar1F5 binds to a conserved region (AGCGK[S/T]) of the helicase motif of bromovirids. The Ilar1R7 primer is located ∼300 nt downstream in a more variable area (GD[K/T][E/K/Q]Q[I/V]P) of the same motif but extensive degeneracy (Table 2) enables it to detect a broad range of species in the family Bromoviridae. No viruses outside this family were detected and although plant material was sometimes amplified, the amplicon was generally a different size. As with the other primers designed during this study, one-step RT-PCR can be used and the PCR products can be sequenced directly (data not shown). The assays described in this report will assist in the characterisation and taxonomic classification of putative members of the Bromoviridae. In RNA1, the Ilar1F5 forward primer is located in the same region as Agdia’s reverse primer; therefore it is possible to sequence more than 700 bp of the genome. Furthermore, the Ilar2F4/Ilar2R9 which detect most ilarviruses allow the amplification of approximately 650 bp. Although not the prime aim of the study, the regions amplified for diagnosis may also be used for phylogenetic studies (Tzanetakis and Martin, 2005; Maliogka et al., 2007).

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