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Journal of Virological Methods 183 (2012) 161– 169

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

jou rn al h om epage: www.elsev ier .com/ locate / jv i romet

acroarray detection of grapevine leafroll-associated viruses

eremy R. Thompsona,∗, Marc Fuchsb, Kael F. Fischerc,d, Keith L. Perrya

Department of Plant Pathology and Plant-Microbe Biology, 334 Plant Science, Cornell University, Ithaca, NY 14850, USADepartment of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY, USADepartment of Pathology, University of Utah, 15 North Medical Drive East, Salt Lake City, UT 84112, USAARUP Laboratories, Salt Lake City, UT 84108, USA

rticle history:eceived 20 March 2012eceived in revised form 17 April 2012ccepted 23 April 2012vailable online 8 May 2012

eywords:LRaVsrrayiagnostics

a b s t r a c t

Grapevine leafroll-associated viruses (GLRaVs) are an emerging group of viruses that represent a sig-nificant threat to the global productivity and sustainability of the grapevine industry. Their control isachieved through the identification and elimination of infected vines, and the use of planting materialderived from virus-tested, certified stocks. As such, much effort has been invested in developing reliablemolecular diagnostic techniques. In this work, we report the development of a macroarray assay for thedetection of the principal GLRaVs. In total 314 70-mer oligonucleotides specific to GLRaV-1, -2, -3, -4, -7,and GLRaV-4 strains 5, 6, 9 and Pr were spotted onto a 11 × 7 cm nylon membrane. Thirty-four grapevinesamples from various origins were tested by the macroarray, RT-PCR and ELISA. Thirty were positivefor virus infection using RT-PCR, 28 by ELISA and 25 by the macroarray. Mixed infections were identi-

ultiplexandom RT-PCR

fied by macroarray in two samples and confirmed by RT-PCR or ELISA. There were a few discrepanciesbetween methods that were most likely due to differences in the sensitivity of detection, and in the caseof the macroarray, limitations in the sequence data available for certain virus species in the design of theoligonucleotides. This work demonstrates the successful application of macroarray methodology usingrandomly primed and sequence-nonspecific amplified cDNAs derived from grapevine total RNA extracts,and provides a proof-of-principal for unbiased multiplex detection using a single robust platform.

Published by Elsevier B.V.

. Introduction

Effective and robust diagnostic methods for the detection oflant viruses are critical for the maintenance of productive andustainable agriculture systems. Currently, the two most commonaboratory methods used in certification schemes are based onither polymerase chain reaction (PCR) or ELISA technologies. Bothave their advantages and disadvantages. PCR is highly sensitive;eing theoretically capable of detecting a single target molecule,ut can give both false positives, because of its sensitivity to con-aminants, and false negatives if the target sequence is not perfectlyomplementary to the primer used to initiate amplification – a rel-tively probable event given the high mutation rates of some RNAiruses (Drake and Holland, 1999). Equally, detection by ELISA isompletely reliant on what degree the antibody employed is able

o recognize virus variants. As a rule, ELISA lacks the sensitivity ofCR by several orders of magnitude (Agindotan and Perry, 2007). Andditional consideration with existing diagnostic methods is their

∗ Corresponding author. Tel.: +1 609 255 0872; fax: +1 607 255 4471.E-mail address: jrt36@cornell.edu (J.R. Thompson).

166-0934/$ – see front matter. Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.jviromet.2012.04.008

ability to be multiplexed thus enabling the simultaneous detectionof a finite number of target molecules. The power of multiplexing inmicroarrays has been amply demonstrated for a variety of animalviruses (Chizhikov et al., 2002; Erlandsson et al., 2011; Klaassenet al., 2004; Kozal et al., 1996; Wang et al., 2002). In plants, the ini-tial adoption of microarrays for virus detection began at a slowerpace with a limited number of virus targets (Boonham et al., 2003;Bystricka et al., 2005; Grover et al., 2010; Pasquini et al., 2008; Weiet al., 2009), although recent reports demonstrate a much broaderapplicability (Tiberini et al., 2010; Zhang et al., 2010) with collec-tions of probes designed to detect up to 52 or more virus species(Bagewadi et al., 2010; Nicolaisen, 2011). The other benefit of anarray system is the theoretical increase in the breadth of detectioninherent in the hybridization kinetics (Naiser et al., 2008; Petersonet al., 2002) between the probe and its target; allowing complemen-tary interactions to occur even when minor mismatches are present(Grover et al., 2010). However, despite the potential of microar-ray detection systems, their use in routine diagnostics is somewhat

restricted due to the technical resources required. More amenableto a standard molecular biological laboratory, macroarraysprovide the diagnostician with a comparable alternative; theonly major equipment required being a thermocycler and a

1 rological Methods 183 (2012) 161– 169

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ybridization oven. The real potential of macroarrays was firstxploited in the detection of fungal, bacterial, and oomycete plantathogens (Fessehaie et al., 2003; Levesque et al., 1998; Lievenst al., 2003). More recently macroarray detection methods haveeen developed for viruses of potato (Agindotan and Perry, 2008;aoka et al., 2010) and solanaceous crops (Perry and Lu, 2010), thus

emonstrating the practicability of crop-specific assays.Globally there is more land devoted to grapevine cultivation

han any other fruit crop, except citrus, with production amount-ng to 39 billion dollars annually (source – http://faostat.fao.org).t least fifty-eight virus species are known to infect grapevine

Martelli and Boudon-Padieu, 2006), of which some of the mostmportant are the grapevine leafroll-associated viruses (GLRaVs)family Closteroviridae) (Martelli et al., 2002). Causing yield lossesrom anywhere between 30 and 68% (Martelli and Boudon-Padieu,006), GLRaVs have been reported, and are considered a serioushreat, in all the major wine growing areas of the world. Primarypread of these viruses is as a result of planting new vineyards withaterial derived from propagated non- or poorly certified bud-ood, while secondary spread, via their mealybug and soft scale

ectors (Fuchs et al., 2009; Tsai et al., 2008, 2010), can occur anyime thereafter. GLRaVs comprise five species: GLRaV-1, -2, -3, -4,nd -7, with GLRaV-4 being an amalgamation of a variety of diver-ent strains, considered until recently separate species, namelyLRaV-4, -5, -6, -9, -De, -Pr and GLRaCV (for more details see (Abouhanem-Sabanadzovic et al., 2012; Thompson et al., 2012)). The

atter six species now are referred to as GLRaV-4 strains 5, 6, 9, De,r, and Car (Martelli et al., 2012). GLRaV-1 and -3 belong to theenus Ampelovirus, sub-group I, GLRaV-4 and its related strains tohe genus Ampelovirus, sub-group II, GLRaV-2 to the genus Clos-erovirus, and GLRaV-7 to the genus Velarivirus (Martelli et al.,012).

GLRaVs present a particular challenge in diagnostics for a num-er of reasons: (i) grapevine tissue is notoriously recalcitrant totandard nucleic acid extraction procedures (Gambino et al., 2008),ii) GLRaVs are mainly phloem-associated, and therefore haveower titers than other more tissue-promiscuous viruses, (iii) virusiters vary depending on the location in the host and the latter’srowth cycle, and (iv) GLRaVs are an emerging group of viruses thatre relatively poorly characterized. Recently, microarray assaysave been developed for the detection of some of these viruses byither prior enrichment of the RNA virus component of the sampley dsRNA isolation (Engel et al., 2010) or by post-hybridisation den-rimer amplification (Abdullahi et al., 2011). In this study we reporthe development of a macroarray assay for the detection of therincipal GLRaVs in grapevine by using, as substrates for labeling,andomly primed sequence-nonspecific amplified complementaryNAs (cDNAs) derived from plant total RNA extracts. This estab-

ishes a proof-of-principal for the unbiased multiplex detectionf most viruses associated with grapevine leafroll disease using aingle robust platform.

. Materials and methods

.1. RNA extraction

Total RNA was extracted from 100 mg of leaf, petiole or barkcrapings using the method described by Gambino et al. (2008),issolved in 50 �l sterile distilled water and its quality and quantityeasured spectrophotometrically, by agarose gel separation and

y using plant RNA ndhB specific primers in RT-PCR as an internalontrol, as previously described (Thompson et al., 2003). The overallteps involved in the processing of the plant material from leaf torray are depicted in Fig. 1.

Fig. 1. Flow chart showing steps in the processing of grapevine material for macroar-ray detection.

2.2. Reverse transcription, cDNA amplification and enzymelabeling

For each sample 400 ng of total plant RNA were reverse-transcribed using 200U M-MLV reverse transcriptase (Promega,Fitchburg, WI, USA) with 1 �M of a random anchored primer(5′-TGGTAGCTCTTGATCANNNNN-3′) (Agindotan and Perry, 2008)

and 1 mM dNTPs in a 20 �l volume, according to the manufac-turer’s instructions. To provide an internal control the reaction wasspiked with 5 ng of a control transcript prepared from a cloned

J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169 163

Table 1Oligonucleotides designed in this study for the macroarray.

GLRaV Genea Probe name Probe sequence (5′–3′)

1 CPd2 LR1 Ca.111 GGAATCGACGTGCCGTGCATAGGGTATGCTCTGAAAAACGGGTCGCTGCGGTTCGGACACGAGATTATATLR1 Ca.112 ATATAATCTCGTGTCCGAACCGCAGCGACCCGTTTTTCAGAGCATACCCTATGCACGGCACGTCGATTCC

1 CPd2 LR1 Cb.113 GACGACGATTTACATTATCTAGTAGATAAATCAACCAGAAATAAACGAATCGAAGGGCTGTATACTGAATLR1 Cb.114 ATTCAGTATACAGCCCTTCGATTCGTTTATTTCTGGTTGATTTATCTACTAGATAATGTAAATCGTCGTC

1 HSP70h LR1 Ha.37 CAGGCGTCGTTTGTACTGTGCCGGCTGAGTATAACTCGTTCAAGCGAAGTTTTTTGGGTGTTGCTTTGGALR1 Ha.38 TCCAAAGCAACACCCAAAAAACTTCGCTTGAACGAGTTATACTCAGCCGGCACAGTACAAACGACGCCTG

1 HSP70h LR1 Hb.67 TTCGGAGGAGGGACATTAGATATATCGTTCATATCAAGGTTTAACAACGTCGTGAGCGTGTTATTTTCGALR1 Hb.68 TCGAAAATAACACGCTCACGACGTTGTTAAACCTTGATATGAACGATATATCTAATGTCCCTCCTCCGAA

1 MT/HEL LR1 La.97 ACTACTCATGAGGCGCAAGGTAAGACATACGAAAACGTGGTCTTGGTGAGACTGAAGAAGGCGGATGATCLR1 La.98 GATCATCCGCCTTCTTCAGTCTCACCAAGACCACGTTTTCGTATGTCTTACCTTGCGCCTCATGAGTAGT

1 RdRp LR1 Ra.7 AACGAGCTTGTTTTGTCTGTAGGCGCACAACGGCGTTCTGGTGGTGCGAATACGTGGTTAGGGAACACTCLR1 Ra.8 GAGTGTTCCCTAACCACGTATTCGCACCACCAGAACGCCGTTGTGCGCCTACAGACAAAACAAGCTCGTT

1 RdRp LR1 Rb.13 ATGAGCTGGATATTTCGAAGTACGACAAGTCTCAAGGTGCGTGCATGAAAGATGTGGAAAGGCTAATCTTLR1 Rb.14 AAGATTAGCCTTTCCACATCTTTCATGCACGCACCTTGAGACTTGTCGTACTTCGAAATATCCAGCTCAT

1 RdRp LR1 Rc.19 ATGGTAAAGGCGGATGCGAAGCCGAAGTTGGATTCAACGCCTTTGCAGCAGTATGTGACTGGGCAAAATALR1 Rc.20 TATTTTGCCCAGTCACATACTGCTGCAAAGGCGTTGAATCCAACTTCGGCTTCGCATCCGCCTTTACCAT

1,3,4 strain 9b RdRp LR139 Ra.1 AAGGAATTGGTGTTGTCTGTCGGCGCACAACGGCGTTCTGGTGGTGCAAACACTTGGATAGGGGACACTGLR139 Ra.2 CAGTGTCCCCTATCCAAGTGTTTGCACCACCAGAACGCCGTTGTGCGCCGACAGACAACACCAATTCCTT

1,3,4 strain 9b RdRp LR139 Rb.3 ATGAACTGGATATCAGTAAGTATGACAAGTCTCAAGGTGCGTGCATAAAGGCATTGGAGTTGGAAGTGTALR139 Rb.4 TACACTTCCAACTCCAATGCCTTTATGCACGCACCTTGAGACTTGTCATACTTACTGATATCCAGTTCAT

1,3,4 strain 9b RdRp LR139 Rc.5 ATGGTGAAAGCAGACGTAAAACCGAAGTTGGATTCAACGCCTTTGCAAGAATTCGCGCCCGGTCAAAACALR139 Rc.6 TGTTTTGACCGGGCGCGAATTCTTGCAAAGGCGTTGAATCCAACTTCGGTTTTACGTCTGCTTTCACCAT

2 CP LR2 Ca.115 AACTTGAGCGGTCTCGTCATAACCGACGCTTCTAGTTTGAATGGTGTCGATAAGAAACTGCTGTCTGCGGLR2 Ca.116 CCGCAGACAGCAGTTTCTTATCGACACCATTCAAACTAGAAGCGTCGGTTATGACGAGACCGCTCAAGTT

2 CP LR2 Cb.117 AACCTTAGCAACCTGGTGATAACCGACGCCTCTAGTCTAAATGGTGTCGACAAGAAGCTTTTATCTGCTGLR2 Cb.118 CAGCAGATAAAAGCTTCTTGTCGACACCATTTAGACTAGAGGCGTCGGTTATCACCAGGTTGCTAAGGTT

2 HSP70h LR2 Ha.83 ACGGTGTGTGTGTACAAGGATGGACGAGTTTTTTCTTTCAAGCAGAATAATTCGGCGTACATCCCTACTTLR2 Ha.84 AAGTAGGGATGTACGCCGAATTATTCTGCTTGAAAGAAAAAACTCGTCCATCCTTGTACACACACACCGT

2 HSP70h LR2 Hb.85 ACGGTGTGCGTGTACAAAGATGGACGAGTTTTCTCGTTCAAGCAGAACAATTCGGCATACATCCCTACTTLR2 Hb.86 AAGTAGGGATGTATGCCGAATTGTTCTGCTTGAACGAGAAAACTCGTCCATCTTTGTACACGCACACCGT

2 HSP70h LR2 Hc.87 ACGGTCTGCGTGTATAAAGATGGTAAGGTTTACTCATTCAAACAAAACAATTCTGCGTACATTCCGACATLR2 Hc.88 ATGTCGGAATGTACGCAGAATTGTTTTGTTTGAATGAGTAAACCTTACCATCTTTATACACGCAGACCGT

2 HSP70h LR2 Hd.89 ACTGTCTGCGTGTACAAGGATGGTAAAGTTTACTCATATAAGCAAAATAACTCCGCGTATATTCCGACGTLR2 Hd.90 ACGTCGGAATATACGCGGAGTTATTTTGCTTATATGAGTAAACTTTACCATCCTTGTACACGCAGACAGT

2 MT/HEL LR2 La.99 TAACCGTGCACGAGGCGCAAGGTGAGACATACGCGCGTGTGAACCTTGTGCGATTTAAGTTTCAGGAGGALR2 La.100 TCCTCCTGAAACTTAAATCGCACAAGGTTCACACGCGCGTATGTCTCACCTTGCGCCTCGTGCACGGTTA

2 MT/HEL LR2 Lb.101 GGTGGATATTCTTCTTTACGAAGCTCCTCCCGGAGGTGGTAAGACGACGACGCTGATAGATTCCTTCTTGLR2 Lb.102 CAAGAAGGAATCTATCAGCGTCGTCGTCTTACCACCTCCGGGAGGAGCTTCGTAAAGAAGAATATCCACC

2 RdRp LR2 Ra.25 AAGTTGATGGTGAAGCGTGACGCTAAGGTGAAGTTAGACTCTTCTTGTTTAACTAAACACAGCGCCGCTCLR2 Ra.26 GAGCGGCGCTGTGTTTAGTTAAACAAGAAGAGTCTAACTTCACCTTAGCGTCACGCTTCACCATCAACTT

2 RdRp LR2 Rb.27 CTTAAGCCTAACATAAAATTTTTTACGGAGATGACTAACAGGGATTTTGCTTCTGTTGTCAGCAACATGCLR2 Rb.28 GCATGTTGCTGACAACAGAAGCAAAATCCCTGTTAGTCATCTCCGTAAAAAATTTTATGTTAGGCTTAAG

2 RdRp LR2 Rc.29 CTCGCGTGAAGGGATGGCAGGTTTTTTACACGTCGGTTAAGAAAGCGCTTTTCAAGAGCGGGTGTTCTCTLR2 Rc.30 AGAGAACACCCGCTCTTGAAAAGCGCTTTCTTAACCGACGTGTAAAAAACCTGCCATCCCTTCACGCGAG

3 CP LR3 Ca.119 ATGGCATTTGAACTGAAATTAGGGCAGATATATGAAGTCGTCCCCGAAAATAATTTGAGAGTTAGAGTAGLR3 Ca.120 CTACTCTAACTCTCAAATTATTTTCGGGGACGACTTCATATATCTGCCCTAATTTCAGTTCAAATGCCAT

3 CP LR3 Cb.121 TTTAGTAAGGCGAGTTTCTTAAAGTACGTTAAGGACGGGACACAGGCGGAATTAACGGGAATCGCCGTAGLR3 Cb.122 CTACGGCGATTCCCGTTAATTCCGCCTGTGTCCCGTCCTTAACGTACTTTAAGAAACTCGCCTTACTAAA

3 HSP70h LR3 Ha.39 TTTTACCCAGACCCGGTTATTGCTGTTATGACTGGGGGGTCAAGTGCTCTAGTTAAGGTCAGGAGTGATGLR3 Ha.40 CATCACTCCTGACCTTAACTAGAGCACTTGACCCCCCAGTCATAACAGCAATAACCGGGTCTGGGTAAAA

3 HSP70h LR3 Hb.43 GCTAGGGCTGTGGAAGTATTCAAAACTGGTCTTGACAACTTTTACCCAGACCCGGTTATTGCTGTTATGALR3 Hb.44 TCATAACAGCAATAACCGGGTCTGGGTAAAAGTTGTCAAGACCAGTTTTGAATACTTCCACAGCCCTAGC

3 HSP70h LR3 Hc.69 GCTAAGGTTTACTGCGATATTTTGGCAGGTAATAGCGGACTGAGACTGGTGGACACTTTAACGAATACGCLR3 Hc.70 GCGTATTCGTTAAAGTGTCCACCAGTCTCAGTCCGCTATTACCTGCCAAAATATCGCAGTAAACCTTAGC

3 MT/HEL LR3 La.103 GACAGTGCATGAAGCACAGGGAAAAACATTCAGTGATGTGGTATTGTTTAGGACGAAGAAAGCCGATGACLR3 La.104 GTCATCGGCTTTCTTCGTCCTAAACAATACCACATCACTGAATGTTTTTCCCTGTGCTTCATGCACTGTC

3 RdRp LR3 Ra.9 AAGGAATTGGTGTTGTCTGTCGGCTCTCAGAGGCGCAGTGGTGGTGCTAACACGTGGTTAGGAAATAGTTLR3 Ra.Pr AACTATTTCCTAACCACGTGTTAGCACCACCACTGCGCCTCTGAGAGCCGACAGACAACACCAATTCCTT

3 RdRp LR3 Rb.15 ATGAACTGGATATCAGTAAGTACGACAAATCTCAGAGTGCTCTCATGAAACAGGTGGAAGAGTTGATACTLR3 Rb.16 AGTATCAACTCTTCCACCTGTTTCATGAGAGCACTCTGAGATTTGTCGTACTTACTGATATCCAGTTCAT

3 RdRp LR3 Rc.21 ATGGTGAAAGCAGACGTAAAACCCAAGTTGGATAATACGCCATTGTCGAAGTACGTAACGGGGCAGAATALR3 Rc.22 TATTCTGCCCCGTTACGTACTTCGACAATGGCGTATTATCCAACTTGGGTTTTACGTCTGCTTTCACCAT

4 CP LR4 Ca.123 GATGAGATAAAGACGGCGCTGGACAACTCCATTGGGTCTTTCGGGTATGAGAACACGCTGAGGCAGTTTGLR4 Ca.124 CAAACTGCCTCAGCGTGTTCTCATACCCGAAAGACCCAATGGAGTTGTCCAGCGCCGTCTTTATCTCATC

4 CP LR4 Cb.125 AAATCTGTGCTTCACATGGTATTCCACCTAACTACTATCCTTACTCTCCAGATTGCCTGCATGTTGATGCLR4 Cb.126 GCATCAACATGCAGGCAATCTGGAGAGTAAGGATAGTAGTTAGGTGGAATACCATGTGAAGCACAGATTT

4 HSP70h LR4 Ha.45 GGAAGGGGAGTTGAAGGTTGTGTTCCTGAAAGTGATACGATATACATACCAACTGTTGTGGGTATAAGGALR4 Ha.46 TCCTTATACCCACAACAGTTGGTATGTATATCGTATCACTTTCAGGAACACAACCTTCAACTCCCCTTCC

4 HSP70h LR4 Hb.57 AGTATAGGACCGGTTTCTGGAGAGAAAGGTAAAACAAGGAGCGTAATTGCACTCGCTTGTATGTTCGTCTLR4 Hb.58 AGACGAACATACAAGCGAGTGCAATTACGCTCCTTGTTTTACCTTTCTCTCCAGAAACCGGTCCTATACT

4 HSP70h LR4 Hc.71 AAAACAAGGAGCGTAATTGCACTCGCTTGTATGTTCGTCTCAGCTCTGGCAAAAATGGCCGTTTCGATAALR4 Hc.72 TTATCGAAACGGCCATTTTTGCCAGAGCTGAGACGAACATACAAGCGAGTGCAATTACGCTCCTTGTTTT

4 P23 LR4 La.105 TATTTTCGTTTGAAGATATGGAGGTTATTCTTTGCTTGTCTAAAGTGGAGACAGACATTCCTGTCGTTGALR4 La.106 TCAACGACAGGAATGTCTGTCTCCACTTTAGACAAGCAAAGAATAACCTCCATATCTTCAAACGAAAATA

4,5,6,9,Prb HSP70h LR4569Pr Ha.31 GCTCTGTTGGAGAAGGATGTGCTGGTCTACAGGGACATAAAGAGATACTTCGGCATGAACAAGTTCAATG

164 J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169

Table 1 (Continued)

GLRaV Genea Probe name Probe sequence (5′–3′)

LR4569Pr Ha.32 CATTGAACTTGTTCATGCCGAAGTATCTCTTTATGTCCCTGTAGACCAGCACATCCTTCTCCAACAGAGC4,5,6,9,Prb HSP70h LR4569Pr Hb.33 GAGAAAGGAAAAACAAGGAGTGTTATTGCCTTGGCGTGCATGTTCGTGTCCGCTTTGGCAAAGATGGCCG

LR4569Pr Hb.34 CGGCCATCTTTGCCAAAGCGGACACGAACATGCACGCCAAGGCAATAACACTCCTTGTTTTTCCTTTCTC4,5,6,9,Prb HSP70h LR4569Pr Hc.35 CGTGTGCTCTGTGCCGGCTGAATACAGTTCGTACATGAGGAATTTTATATTCCAAGGTTGTAATTTGGCT

LR4569Pr Hc.36 AGCCAAATTACAACCTTGGAATATAAAATTCCTCATGTACGAACTGTATTCAGCCGGCACAGAGCACACG4 strain 5 HSP70h/p60 LR5 55a.107 ATGGCATTGTCTGCGACTAGCACCTACGTTCGGGTGTCGAAGAGCGATGAAGGGTTCGTCAATCTGCTGA

LR5 55a.108 TCAGCAGATTGACGAACCCTTCATCGCTCTTCGACACCCGAACGTAGGTGCTAGTCGCAGACAATGCCAT4 strain 5 CP LR5 Ca.127 GAGCTCTCTGGAACTTGGGGAAATCAAAAGGGATATCTGAGAGTGATAAGCATCAGGTACAGTTCTTGAT

LR5 Ca.128 ATCAAGAACTGTACCTGATGCTTATCACTCTCAGATATCCCTTTTGATTTCCCCAAGTTCCAGAGAGCTC4 strain 5 CP LR5 Cb.129 GACAATTCTATAGGGTCTTTCGGTTACGAAAACACTCCTAGACAATTTGGAAGAGCATTCACGGCAGCGA

LR5 Cb.130 TCGCTGCCGTGAATGCTCTTCCAAATTGTCTAGGAGTGTTTTCGTAACCGAAAGACCCTATAGAATTGTC4 strain 5 HSP70h LR5 Ha.47 GGCAGAGGCGTGGACGGTTGTGTTCCTGAGAGCGACACGGTGTATATACCGACGGTGGTTGGTATAAGAA

LR5 Ha.48 TTCTTATACCAACCACCGTCGGTATATACACCGTGTCGCTCTCAGGAACACAACCGTCCACGCCTCTGCC4 strain 5 HSP70h LR5 Hb.59 TGTGTTCTGTACCTGCTGAGTACAGCTCCTACATGAGGAATTTCATTTTCCAAGGTTGTAGTTTGGCTAA

LR5 Hb.60 TTAGCCAAACTACAACCTTGGAAAATGAAATTCCTCATGTAGGAGCTGTACTCAGCAGGTACAGAACACA4 strain 5 HSP70h LR5 Hc.73 TAAAACCCAAGTTCGAGGTCATAGTGAAAAACTGGTCAGCTTACATTGGTCCATCTTCTGGAGAGAAAGG

LR5 Hc.74 CCTTTCTCTCCAGAAGATGGACCAATGTAAGCTGACCAGTTTTTCACTATGACCTCGAACTTGGGTTTTA4 strain 6 HSP70h LR6 Ha.49 GGACGAGGCGTGGATGGTTGTGTTCCAGAGAGCGACACCATCTACATACCGACGGTTGTTGGTATCAGGA

LR6 Ha.50 TCCTGATACCAACAACCGTCGGTATGTAGATGGTGTCGCTCTCTGGAACACAACCATCCACGCCTCGTCC4 strain 6 HSP70h LR6 Hb.61 AGCGAAGATGGCTGTTAAGATGACCGGTAGTGCTGTGGTTTTGTCGGTGTGTTCTGTGCCCGCGGAATAT

LR6 Hb.62 ATATTCCGCGGGCACAGAACACACCGACAAAACCACAGCACTACCGGTCATCTTAACAGCCATCTTCGCT4 strain 6 HSP70h LR6 Hc.75 GGTATGAACAAGTTCAACGCTGAAGGTTACAGAAAGAAACTGAAGCCGAAATTTGAGGTGGTAGTTAAAA

LR6 Hc.76 TTTTAACTACCACCTCAAATTTCGGCTTCAGTTTCTTTCTGTAACCTTCAGCGTTGAACTTGTTCATACC7 HSP70h LR7 Ha.41 TATCAGAAATCTCTCTTGAAGGAGATAGGTTTTTCTGTCAACCAAATTGTGAATGAACCTAGTGCCGCCG

LR7 Ha.42 CGGCGGCACTAGGTTCATTCACAATTTGGTTGACAGAAAAACCTATCTCCTTCAAGAGAGATTTCTGATA7 HSP70h LR7 Hb.51 ACTACTTTTAGTACTGTATCAGCTTTAATCGATGACAAATTTATTGAATTAAATATGAATGGATCACCAT

LR7 Hb.52 ATGGTGATCCATTCATATTTAATTCAATAAATTTGTCATCGATTAAAGCTGATACAGTACTAAAAGTAGT7 HSP70h LR7 Hc.77 ATAGGTTCTGCCGCGAAACTTGTTCATCTTAAGTCGGACAAATATACTATTTACTATGATTTGAAACGTT

LR7 Hc.78 AACGTTTCAAATCATAGTAAATAGTATATTTGTCCGACTTAAGATGAACAAGTTTCGCGGCAGAACCTAT7 HSP70h LR7 Hd.91 AAATCAAATCTATACAACCTGGAAGACTAAACATTTCTGTACCGGCCGATTATTCAAATTTGCAAAGAGG

LR7 Hd.92 CCTCTTTGCAAATTTGAATAATCGGCCGGTACAGAAATGTTTAGTCTTCCAGGTTGTATAGATTTGATTT7 HSP70h LR7 He.93 ATGGGATCGGCACTGTAAGAAAGTTTTTACCAGTTAAATCCTTGATTTATTACTATATAAAGAACTTAGT

LR7 He.94 ACTAAGTTCTTTATATAGTAATAAATCAAGGATTTAACTGGTAAAAACTTTCTTACAGTGCCGATCCCAT7 HSP70h LR7 Hf.95 TGAATTCTTTGAATTTTGAAAGAATAAGAGACAAATTAAAACCTAAATACAATACAATATTTCAGGACAA

LR7 Hf.96 TTGTCCTGAAATATTGTATTGTATTTAGGTTTTAATTTGTCTCTTATTCTTTCAAAATTCAAAGAATTCA4 strain 9 HSP70h LR9 Ha.53 GGCAGAGGTGTTGAAGGTTGTGTTCCGGAGAGTGACACGGTGTACATACCGACAGTGGTCGGCATAAGAA

LR9 Ha.54 TTCTTATGCCGACCACTGTCGGTATGTACACCGTGTCACTCTCCGGAACACAACCTTCAACACCTCTGCC4 strain 9 HSP70h LR9 Hb.63 TCGTACATGAGGAACTTTATCTTCCAAAGCTGTAGTTTGGCTAAAATTCAGGTTCAAGCAGTGGTGAATG

LR9 Hb.64 CATTCACCACTGCTTGAACCTGAATTTTAGCCAAACTACAGCTTTGGAAGATAAAGTTCCTCATGTACGA4 strain 9 HSP70h LR9 Hc.79 TAGGGCCAACGTCTGGTCAAAAGGGAAAGACAAGGAGTGTCATTGCTTTGGCCTGCATGTTTGTGTCGGC

LR9 Hc.80 GCCGACACAAACATGCAGGCCAAAGCAATGACACTCCTTGTCTTTCCCTTTTGACCAGACGTTGGCCCTA4 strain 9 RdRp LR9 Ra.11 AAAAGTTTTTCTGCCGAAATCTTCTCTCAAAGGCGAACTGGTGCTGCAAACACTTGGATAGGGGACACTG

LR9 Ra.12 CAGTGTCCCCTATCCAAGTGTTTGCAGCACCAGTTCGCCTTTGAGAGAAGATTTCGGCAGAAAAACTTTT4 strain 9 RdRp LR9 Rb.17 ACGAAGTAGACATATCGAAGTATGACAAATCTCAAAACACCTTCACAAAGGCATTGGAGTTGGAAGTGTA

LR9 Rb.18 TACACTTCCAACTCCAATGCCTTTGTGAAGGTGTTTTGAGATTTGTCATACTTCGATATGTCTACTTCGT4 strain 9 RdRp LR9 Rc.23 ATGGTTAAATCGGATGTTAAATTTAAAGGAGACTCCGAAGCGTTGGAAGAATTCGCGCCCGGTCAAAACA

LR9 Rc.24 TGTTTTGACCGGGCGCGAATTCTTCCAACGCTTCGGAGTCTCCTTTAAATTTAACATCCGATTTAACCAT4 strain Pr CP LRPr Ca.131 CTCTTTAACGTTGCTATACCGGGCGTTGTTGCCGGTAAGCACAAAGTTATTGGAGCCAAGGCTTTGTGGG

LRPr Ca.132 CCCACAAAGCCTTGGCTCCAATAACTTTGTGCTTACCGGCAACAACGCCCGGTATAGCAACGTTAAAGAG4 strain Pr CP LRPr Cb.133 TGGTGGCACACGACGAGATCAAAACTACTCTGGACAACTCCATCGCGGCGTTTGGGTATGAAAATACACT

LRPr Cb.134 AGTGTATTTTCATACCCAAACGCCGCGATGGAGTTGTCCAGAGTAGTTTTGATCTCGTCGTGTGCCACCA4 strain Pr HSP70h LRPr Ha.55 GGACGAGGCGTCGATGGTTGTGTTCCTGAGTCGGACACAATCTACATACCGACGGTGGTTGGTGTGAGGC

LRPr Ha.56 GCCTCACACCAACCACCGTCGGTATGTAGATTGTGTCCGACTCAGGAACACAACCATCGACGCCTCGTCC4 strain Pr HSP70h LRPr Hb.65 TCTGTGCCGGCTGAGTACAGTTCTTACATGAGGAATTTCATTTTCCAAGGTTGTAAGTTGGCTAAGATAC

LRPr Hb.66 GTATCTTAGCCAACTTACAACCTTGGAAAATGAAATTCCTCATGTAAGAACTGTACTCAGCCGGCACAGA4 strain Pr HSP70h LRPr Hc.81 TTCTTGTGTACAGGGATATAAAACGATACTTCGGGATGAATAAATTCAATGCTGATTCTTACATGAAGAA

LRPr Hc.82 TTCTTCATGTAAGAATCAGCATTGAATTTATTCATCCCGAAGTATCGTTTTATATCCCTGTACACAAGAA4 strain Pr MT/HEL LRPr La.109 CACGGTTGCTGAAAGTCAAGGAGAGACATACTCTAGAGTGGCTTTGGTTAGGACCAAGGCAGCGGACGAT

LRPr La.110 ATCGTCCGCTGCCTTGGTCCTAACCAAAGCCACTCTAGAGTATGTCTCTCCTTGACTTTCAGCAACCGTG

a CP – coat protein, HSP70h – heat shock protein 70 homolog, MT/HEL – methyltransferase/helicase, RdRp – RNA-dependent RNA polymerase. oligon

c(cf1Etfo0

b This indicates the oligonucleotide is one of the ‘Pol’ and ‘HSP’ group of ‘generic’

omplementary DNA fragment derived from a West Nile VirusWNV) NS1 protein (kindly provided by R. Renshaw). This 1057 bpDNA fragment corresponds to nucleotide positions 2472–3528rom GenBank sequence NC 009942. The cDNA in vector TriEx-

(Novagen) and was transcribed with T7 RNA polymerase (Newngland Biolabs, Ipswich, MA, USA). Once the reverse transcrip-

ion reaction was complete, 0.5 �l was used as the templateor PCR amplification using IQ supermix (BioRad) with 0.2 �Mf the above random anchored primer in combination with.8 �M of the anchor primer (5′-AGAGTTGGTAGCTCTTGATC-3′).

ucleotide probes.

Thermocycler conditions were: 94 ◦C for 5 min; 94 ◦C for 30 s, 55 ◦Cfor 1 min, 72 ◦C for 2 min (40×); 72 ◦C for 7 min, after which theamplified products were checked on a 1% agarose gel, purifiedusing QIAquick PCR purification kit (Qiagen) and eluted in 25 �lsterile distilled water. Four hundred ng of the purified amplifiedcDNA, having been quantified spectrophotometrically, was dena-

tured by boiling in a total volume of 20 �l of sterile distilled waterfor 5mins, and placed immediately on ice. The denatured cDNA waslabeled with alkaline phosphatase by adding the components of theAlkPhos Direct Labeling and Detection System with CDP-Star (GE

J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169 165

Fig. 2. Schematic of the position, relative to the viral genome, of the oligonucleotides used for the macroarray. In each panel, the genome of the virus (GLRaV-1, -2, -3, -4, -4strain 5, -4 strain 6, -7, -4 strain 9 and -4 strain Pr) is depicted above with each gene or domain shown as a gray box. In the lower half of each panel, the genome is depictedas a single line with 5′ to 3′ orientation, and the relative position of probes is shown as dots colored red for probes designed in this work, or black for probes designed byEngel et al. (2010) or by Bagewadi et al. (2010) for a universal plant virus microarray. Below the genome, the area of the sequence coverage is shown as black lines, witht the NCp RNA pC domP

Hla

2

v7(dImfdsGseaos

he number at the left of each line indicating the number of sequences available in

rotein 70 homolog, MT/HEL – methyltransferase/helicase, RdRp – RNA-dependent

ircle, square and triangle refer to the positions in the MT/HEL gene of the conservedositions of generic probes; LR139(‘Pol’) and LR4569PR(‘HSP’) are not included.

ealthcare): 20 �l reaction buffer, 16 �l sterile distilled water, 4 �labeling reagent, and 4 �l crosslinker, and incubating for 30 mint 37 ◦C.

.3. Oligonucleotide probes and membrane printing

Probes for GLRaVs were designed both in the plus and minusiral sense and were derived from three sources; (1) in-house0-mers, (2) 70-mer oligonucleotides designed for a microarrayTable S1) (Engel et al., 2010), and (3) 60-mer oligonucleotidesesigned for a microarray (Bagewadi et al., 2010) (Table S2).

n-house probes were designed by visual selection (based on theost conserved regions with the most sequence information)

rom consensus sequences obtained by aligning all available NCBIatabase sequences for each virus species and GLRaV-4 relatedtrains assuming GLRaV-4, GLRaV-4 strain 5, GLRaV-4 strain 6,LRaV-4 strain 9 and – GLRaV-4 strain Pr are to be considered aingle species (Abou Ghanem-Sabanadzovic et al., 2012; Thompson

t al., 2012) (Table 1, Fig. 2). There were 12 exceptions to thispproach. Six probes designated LR139 (termed the ‘Pol’ group ofligonucleotides) whose sequences were derived from conservedequence stretches present in the aligned RNA-dependent RNA

BI database for probe design. Gene names: CP – coat protein, HSP70h – heat shockolymerase, and proteins (p followed by a number indicating the size in kilodaltons.ains of the methyltransferase, AlkB and helicase, respectively. Black box – protease.

polymerase genes of GLRaV-1, -3 and -9 and were made with theidea of producing generic ampelovirus specific probes (Table 1,footnote ‘b’). However, instead of using a cross species consensussequence, a sequential composite of each species sequence (orconsensus in the case of GLRaV-1) was constructed consistingof 23, 23 and 24 nts respectively for each virus. Six additionalprobes designated LR4569PR (termed the ‘HSP’ group of oligonu-cleotides) were designed in a similar fashion using the mostconserved region of the heat shock protein homolog (HSP70h)gene upon aligning all sequences available for GLRaV-4 and relatedGLRaV-4 strains 5, 6, 9 and Pr (Table 1). Control probes were: theribosomal RNA specific rRNA-1, -5 and -6 as described previously(Agindotan and Perry, 2007), ST-1 trimer (5′-CACGGCGATTTCGCA-GTTTACACGGCGATTTCGCAGTTTACACGGCGATTTCGCAGTTTA-3′; a gift of A. Levesque) and WNV-1 (5′-ATGGCGAT-ATCCACTGGGTGTGCCATAAACATCAGCCGGCAAGAGCTGAGATG-TGGAAGTGGAGTGTTCA-3′), and WNV-2 (5′-CCGGCAAGAG-CTGAGATGTGGAAGTGGAGTGTTCATACACAATGATGTGGAGGCTT-

GGATGGACCGGTAC-3′) specific to the amplicon derived fromthe internal control WNV transcript introduced at the reversetranscription stage (described above). Most oligonucleotideswere spotted at a final concentration of 20 �M, while the rRNA

166 J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169

Fig. 3. Layout of the oligonucleotide positions on the macroarray. The outer left-hand column and upper row show the array co-ordinate numbers. Reference controloligonucleotides ‘ST1/Rib’ are spotted along the top and sides of the array; an additional column of these control probes is spotted in the center of the membrane. Otherc the ao es ans (5, 6, 9l

pdzF

ontrol probes (Rib1, 5, 6, WNV1/2) are spotted in columns on the right side ofligonucleotides are printed in two blocks, grouped as plus (virion) sense sequencequences from individual GLRaV species (1, 2, 3, 4, 7) and GLRaV-4 related strains

ast four columns of each block.

robes were at 2 �M. The printing procedure was carried out asescribed previously (Agindotan and Perry, 2007). The organi-ation of the probes on the nylon membrane was as shown inig. 3.

rray, and all of these are labeled at the bottom of the figure. The virus-specificd minus (complementary) sense sequences. The columns of probes correspond to, Pr). Additional virus generic oligonucleotides (Pol/HSP, UT5, AMP, CLO) are in the

2.4. Hybridization and detection

Used multiple (10–20) times the membranes were strippedprior to use by incubating at 60 ◦C in 0.5% (wt/vol) sodium dodecyl

J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169 167

Table 2List of grapevine samples and their infection status based on different diagnostic methods.

Vine accession/isolatea Array ELISAb RT-PCR

InfectedVitis vinifera cv. Ahmed Sal Y163 GLRaV-1 GLRaV-1 GLRaV-1V. vinifera cv. Chaouch rose Y206 GLRaV-2 GLRaV-2 GLRaV-2V. vinifera cv. Raziki Y285 GLRaV-3 GLRaV-3 GLRaV-3Vitis hybrid cv. Estellat GLRaV-3, -4 strain 5 GLRaV-3, -4/9, -4 strain 5, -4 strain 6 GLRaV-3, -4 strain 5V. labrusca cv. Concordc GLRaV-3 GLRaV-3 GLRaV-3V. vinifera cv. Italia 3 GLRaV-3, -4, -4 strain 5, -7, -4 strain Pr Negative GLRaV-3, -4 strain 5, -7d

V. vinifera cv. Tannat Negative GLRaV-4/9, -4 strain 5 GLRaV-4 strain 9V. vinifera cv. Koudsi Y252 Negative GLRaV-4/9 GLRaV-4V. vinifera cv. Koussan Y253 Negative GLRaV-4/9 GLRaV-4V. vinifera cv. Molinera 11648 GLRaV-4, -4 strain 5 GLRaV-4/9, -4 strain 5, -4 strain 6 GLRaV-4 strain 5V. vinifera cv. White Emperor Y217 GLRaV-4 strain 5 GLRaV-4 strain 5 GLRaV-4 strain 5V. vinifera cv. Thompson Seedless 34924 GLRaV-4 strain 5e GLRaV-4/9 GLRaV-4V. vinifera cv. Kandari Y243 Negative GLRaV-7 NegativeV. vinifera cv. Pinot noir 23 Negative Negative GLRaV-7V. vinifera cv. Helena LR118 Negative Negative GLRaV-4 strain 9V. vinifera cv. Cabernet 34964 GLRaV-4 strain 9 GLRaV-4/9 GLRaV-4 strain 9

HealthyV. vinifera cv. Riesling N90 Negative Negative NegativeV. vinifera cv. Cabernet franc TJBf Negative Negative nt

a Samples in boldface are those for which array results are shown in Fig. 4.b Serological reagents specific to GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, strain 5, GLRaV-4 strain 6, or GLRaV-7 are used as well as generic antibodies to GLRaV-4/9 (=GLRaV-4,

GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-7 and GLRaV-4 strain 9).c Represents sixteen Concord vines that all tested positive for GLRaV-3.d Amplicon not sequenced.e This designation of GLRaV-4 strain 5 is based on the hybridization of two of 8 probes with design based on the sequence of ‘GLRaV-5′; in most assays, extracts hybridize

to a majority of the probes for a given species.f Cabernet franc results are for two vines tested.

sftLf0tawNDoawdUcwca

2

nG(afttaCr

ulfate (SDS) for 1 h followed by a rinse in 0.1 M Tris–HCl pH 8.4or 5 min. Pre-hybridization for 1 h was done in 10 ml of hybridiza-ion buffer (supplied with the AlkPhos Direct kit, GE Healthcare,ittle Chalfont, UK) at 55 ◦C, after which all the labeled PCR productrom the AlkPhos labeling reaction (64 �l) and an internal control of.5 ng AlkPhos labeled ST-1 trimer were added to 5 ml of hybridiza-ion buffer and applied to the membrane. Hybridization was donet 55 ◦C overnight. Membranes were washed twice in a primaryash buffer (2 M urea, 0.1% SDS, 50 mM sodium phosphate, 150 mMaCl, and 0.2% [wt/vol] blocking agent supplied with the AlkPhosirect kit) at 55 ◦C for 15 min each, followed by washing in a sec-ndary wash buffer (50 mM Tris, 100 mM NaCl, pH 10.0) for 10 min,t room temperature on a shaker. The membranes were incubatedith CDP-star chemiluminescent (GE Healthcare) reagent for 5 min,rained, and viewed both in a Biospectrum® 500 Imager (UVP,pland, CA, USA) for 10–45 min and by exposing to Chemilumines-ence Bio Max film (Kodak, Rochester, NY, USA) overnight. Samplesere only considered positive for a virus if more than one oligonu-

leotide spotted pair gave a signal. Membranes were stored moistt 4 ◦C sealed in a plastic bag.

.5. ELISA and RT-PCR

ELISA was carried out using antibodies to GLRaV-5 (AC Diag-ostics, Fayetteville, AR, USA) and GLRaV-1, GLRaV-2, GLRaV-3,LRaV-6, or GLRaV-7 as well as generic antibodies to GLRaV-4/9

=GLRaV-4, GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-4 strain 9nd GLRaV-7) (Bioreba, Reinach, Switzerland) according the manu-acturer’s instructions. RT-PCR was done with primer pairs specifico each virus using the same conditions described in the publication

hat initially reported them (Table S3). Reverse transcription (RT)nd PCR were done with M-MLV reverse transcriptase (Invitrogen,arlsbad, CA, USA) and iQTM Supermix (Biorad, Hercules, CA, USA),espectively.

3. Results and discussion

Thirty-four vine samples were analyzed by macroarray analy-sis, ELISA and RT-PCR. Results from the three methods were forthe most part consistent, with 25, 28 and 30 samples positive usingthe macroarray, ELISA and RT-PCR, respectively (Table 2). Examplesof macroarray results are shown for vines with GLRaV-1 (Fig. 4a),GLRaV-2 (Fig. 4b), GLRaV-3 (Fig. 4c) and GLRaV-4 strain 5 (Fig. 4d).Signals in the macroarray were often faint, but clearly distinguish-able from background. For samples identified by RT-PCR and ELISAas singly infected by GLRaV-1, -2, -3 and GLRaV-4 strain 5 the signalsfrom the probes were relatively strong and unambiguous. GLRaV-4and GLRaV-4 strains 5, 6, and 9 are all closely related; thus, crosshybridization among the probes corresponding to their sequencesis expected. Vines such as V. vinifera cv. Molinera 11648 might bebest described as being infected by GLRaV-4-like virus(es) ratherthan having multiple virus species.

Although results from the macroarray analysis were consistentwith those of the other methods for 25 of the 33 vines, some dis-crepancies were apparent. The most obvious difference betweenthe methods was for six vines testing negative for virus by macroar-ray, but not by ELISA and/or RT-PCR. Three vines (V. vinifera cv.Tannat, V. vinifera cv. Koudsi Y252, V. vinifera cv. Koussan Y253)tested positive by ELISA for the GLRaV-4 viral complex and specif-ically GLRaV-4 by RT-PCR, but were negative with the macroarray.Similarly, two vines (V. vinifera cv. Kandari Y243 and V. vinifera cv.Pinot noir 23) that tested positive for the presence of GLRaV-7,tested negative with the macroarray. The rationale for these dif-ferences is best explained as being due to a combination of lowvirus titer and/or the limited sequence information available foroligonucleotide design. The limited sequence information available

is apparent upon assessing the distribution of the oligonucleotidesdesigned, in particular for GLRaV-4, GLRaV-4 strain 6 and GLRaV-7(Fig. 2); this was dictated by the amount of sequence data availablein the database at the time this work began in 2010. For all three

168 J.R. Thompson et al. / Journal of Virological Methods 183 (2012) 161– 169

Fig. 4. Microarray results for samples (a) V. vinifera cv. Ahmed Sal Y163, (b) V. vinifera cv. Chaouch rose Y206, (c) V. vinifera cv. Raziki Y285, (d) V. vinifera cv. White EmperorY217, (e) V. vinifera cv. Italia 3, (f) healthy control – V. vinifera cv. Cabernet Franc TJB1-1. Numbering and lettering below each circled column show the oligonucleotide typet bes spa

vwttG42lff

mh35wMbs

tot

hat was detected as binding to the chemiluminescent target: numbers refer to prond Amp refers to probes UPVM 271–294 (Table S1).

iruses, just the N-terminal part of the HSP70h gene was covered,ith GLRaV-4 having some additional sequence data available for

he coat protein (CP) gene and downstream regions. In the past year,here has been a flourish of sequence information for GLRaV-4 andLRaV-4 strain 6 (Abou Ghanem-Sabanadzovic et al., 2012), GLRaV-

strain 5 (Thompson et al., 2012) and GLRaV-7 (Al Rwahnih et al.,012; Jelkmann et al., 2012) providing full-length or close to full-

ength sequences. This increased sequence information will allowor a less limited, genome-wide selection of oligonucleotides in theuture.

The macroarray revealed samples which appeared to haveixed infections, namely V. vinifera cv. Italia 3 (Fig. 4e), Vitis

ybrid cv. Estellat and V. vinifera cv. Molinera 11648 for GLRaV-, GLRaV-4 strains, and GLRaV-7; GLRaV-3 and GLRaV-4 strain; and GLRaV-4 and GLRaV-4 strain 5, respectively. All virusesere confirmed by RT-PCR, except for GLRaV-4 in V. vinifera cv.olinera 11648 – the array results for which can be explained

y possible cross-hybridization of the oligonucleotides betweentrains.

V. vinifera cv. Cabernet 34964 was faintly positive for GLRaV-9 inhe macroarray and RT-PCR, though negative in ELISA. Examinationf the array results (Fig. S1) shows that though the sample is posi-ive for GLRaV-9, the method is at the limits of detection. The visual

ecific to the GLRaV species, ‘HSP’ and ‘Pol’ refer to probes 31–36 and 1–6 (Table 1),

assessment of positive signals was facilitated by three factors: (i)all oligonucleotide probes were spotted in adjacent paired repli-cates; thus positive signals are observed in pairs, (ii) all the probesfor a single virus were spotted sequentially; thus the presence ofviral sequences often results in successive signals in a column,and (iii) both plus and minus-sense oligonucleotide probes werespotted. The latter proves to be useful in situations of low titerwherein signal intensities are at the threshold of detection, becausehybridization can sometimes be observed with a minus sense probebut not with the positive sense probe or vice versa. As a general-ization, in this work minus sense oligonucleotides hybridized morecommonly and with greater intensity than their plus strand coun-terparts. The reason for this is not fully understood, but has beenreported elsewhere in grapevine infecting viruses (Giampetruzziet al., 2012; Grover et al., 2010) and could be attributed to a polaritybias in the generation of siRNAs (Pantaleo et al., 2010).

This work clearly demonstrates the successful application ofmacroarray technology to the detection of some of the most recal-citrant plant viruses present in cultivated crops allowing further

refinements to the methodology and the expansion of the arrayto include all significant grapevine infecting viruses – a processthat is already underway in our lab. Rather than providing a sin-gle diagnostic approach, we hope this method can complement

rologi

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J.R. Thompson et al. / Journal of Vi

ther molecular detection methods by providing the elementsf multiplexing and detection breadth to any future certificationcheme. The relative simplicity and robustness of the methodologyepresents a diagnostic platform that will be accessible to mostolecular biology laboratories. The only aspects that would require

xtra consideration on whether to adopt this method or not woulde the time required to obtain results (from 2 to 3 days) and theost, which will vary and be largely a function of design and thenitial purchase of the oligonucleotides.

cknowledgements

Thanks to Laura Miller, Heather McLane, Rosemary Cox andave MacUmber for their support in the lab. We are indebted tors. Maher Al Rwahnih and Adib Rowhani of Foundation Plantervices, University of California at Davis, Dr. Olivier Lemaire,NRA, Colmar, France, and Dr. Carole Balmelli, Agroscope, Chan-ins, Switzerland for providing some of the samples tested. Thisork was supported in part by funding from the U.S. Department

f Agriculture Animal Plant Health Inspection Service as part of theational Clean Plant Network program and by Agriculture and Foodesearch Initiative Competitive Grants no. 2009-55605-05184 and009-55605-05023 from the USDA National Institute of Food andgriculture.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.jviromet.2012.04.008.

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