virus discovered by deep sequencing

8/13/2019 Virus Discovered by Deep Sequencing

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Virus ResearchVolume 163, Issue 1,January 2012, Pages 262268

A new grapevine virus discovered by deep sequencing of virus- andviroid-derived small RNAs in Cv Pinot gris

Annalisa Giampetruzzia, 1, Vahid Roumia, 1, Roberta Robertoa, Umberto Malossinib, Nobuyuki Yoshikawac, Pierfederico La Nottea, Federica Terlizzid,
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Rino Credid, Pasquale Saldarellia, , aDipartimento di Biologia e Chimica Agroforestale ed Ambientale, Universitdegli Studi di Bari ed Istituto di Virologia Vegetale

del CNR UOS-Bari, via Amendola 165/A, 70126 Bari, Italy

bFondazione Edmund MachIASMA, via E. Mach 138010 San Michele a/A, Italy cFaculty of Agriculture, Iwate University, Morioka 020-8550, Japan dDipartimento di Scienze e Tecnologie Agroambientali, Universitdi Bologna, Viale Fanin, 40, 40127 Bologna, Italia http://dx.doi.org/10.1016/j.virusres.2011.10.010,How to Cite or Link Using DOI Permissions & Reprints

Abstract

Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris(PG) in

the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-,

ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not

symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and

viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated

virus(GRSPaV), two viroids Hop stunt viroid(HSVd) and Grapevine yellow speckle viroid 1(GYSVd1),

the marafivirusesGrapevine rupestris vein feathering virus(GRVFV) and Grapevine Syrah virus 1

(GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that

of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it

grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat proteingene. However, molecular differences with GINV are wide enough to warrant classification of the virus

in question as a new species, for which the provisional name of Grapevine Pinot gris virus(GPGV) is

proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from

three different cultivars did not allow to clearly associating the virus to the observed symptoms.

Highlights

Unknown viral symptoms were observed in cv. Pinot gris. Small RNAs from grapevine tissue weresequenced by Illumina technology. Viruses and viroids infecting Pinot grisplants and properties of

their viral small RNAs were described. De novo assembling of small RNAs allowed assembling the

genome of a new virus. This new virus is taxonomically related to trichoviruses. Limited field

survey for the presence of this virus did not clearly associate the virus to the observed symptoms.

Keywords

Grapevine; Virus; Deep sequencing;
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Contig; Trichovirus

1. Introduction

Symptoms resembling those of a viral disease, i.e. chlorotic mottling, puckering and deformation of the

leaves, reduced yield and low quality of the berries have been observed in 2003, in plants of cv Pinot

gris in vineyards of Trentino (northern Italy) (Fig. 1A and B). Successively (2009) similar symptoms

were described on the cvs Traminerand Pinot noir. Particularly on Traminer, symptoms were more

accentuated, consisting in stunting and significant losses in production (Fig. 1C and D and

unpublished information). No virus particles were detected with the electron microscope in dips from

symptomatic PG leaves and no visible reactions were obtained with ELISA using commercial kits

(Agritest, Italy) toGrapevine fanleaf virus(GFLV),Arabis mosaic virus(ArMV), Strawberry latent

ringspot virus(SLRSV),Grapevine leafroll-associated virus-1 (GLRaV-1), -2 (GLRaV-2) and -3

(GLRaV-3), Grapevine virus A(GVA) and Grapevine virus B(GVB) (unpublished results). Equally

negative were RT-PCR assays using specific primers for the above reported ampelo- and vitiviruses

and degenerate primers for the detection of members of the three subgroups of the

genus Nepovirus(Digiaro et al., 2007).
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Fig. 1. Chlorotic mottling and leaf deformations in the cvs Pinot gris(A and B) and Traminer(C). Shoot stunting in

cv Tramineris shown in panel (D).

Figure options

Deep sequencing is a powerful technology that provides rapid and exhaustive information on the

infectious agents (viruses and viroids) present in plant tissues (Kreuze et al., 2009andWu et al.,

2010). Therefore, this technology is being increasingly used for the quick identification of viruses

replicating in plant tissues, either starting from the analysis of small interfering RNA (siRNAs)

populations (Kreuze et al., 2009), or from sequenced libraries of fragmented double-stranded RNAs

(dsRNAs) of viral origin (Al Rwahnih et al., 2009andCoetzee et al., 2010), extracted from infected

tissues.

With a study of a vine of cv. Pinot Noir clone ENTAV 115, we have recently experienced the high

potentiality of deep sequencing, and have described the virome[sensuCoetzee et al. (2010)]of the

plant (Pantaleo et al., 2010). Thus, we have now deep sequenced and screened the small RNA

population of a symptomatic and a symptomless PG vine, identifying, among other disease agents, a

putatively new virus phylogenetically related to the trichovirus Grapevine berry inner necrosis

virus(GINV;Yoshikawa et al., 1997andKunugi et al., 2000), for which the provisional name

of Grapevine Pinot gris virus(GPGV) is proposed.

2. Materials and methods

2.1. Small RNA purification and sequencing

Leaves and petioles were collected in July 2010 from a symptomatic (plant S1+) and a symptomless

(plant S2) PG vine and stored at 4 C for 5 days before using for total RNAs extraction. This was

carried out as described byPantaleo et al. (2010)and the low molecular weight RNA fraction (LMW-

RNA) was isolated by polyethylenglycol precipitation (Hamilton and Baulcombe, 1999). Small RNAs

were separated by polyacrylamide gel electrophoresis and recovered from the gel as described by Lu

et al. (2007).Libraries of small RNA populations from both vines were prepared for sequencing with an

Illumina Genome Analyzer II (Institute of Applied Genomics, University of Udine, Italy) following the

protocol ofLu et al. (2007),which generated 3644 bp sequences. Adaptors, ribosomal and transfer

RNA sequences were removed by the UEA siRNA toolkit (Moxon et al., 2008) and the small RNA

sequences between 18 and 26 nt in size were initially screened for homology to known viruses by

local BLASTN search against the GenBank Virus Reference Sequence database

(RefSeq;http://www.ncbi.nlm.nih.gov/). Subsequently, libraries were searched with the SOAP (Short

Oligonucleotide Alignment Program;Li et al., 2010) packages of software against reference

sequences retrieved from the RefSeq database. Total small RNAs were then assembled into larger

contigs using the Velvet Software 0.7.31 (Zerbino and Birney, 2008)with a k-mer of 17.

GRSPaV, HSVd and GYSVd1 genomic RNAs were reconstructed by aligning and assembling

contigson database-retrieved sequences, using the DNA Strider software 1.46 (Marck, 1988).

Consensus sequences were finally obtained by the SOAPaligner software, which aligns small RNAs to
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a reference genome, allowing a tolerance of two mismatches. Nucleotide polymorphisms having a

frequency 80% and a minimum coverage of five valid small RNAs, as calculated by MapView (Bao et

al., 2009), were allowed in the consensus sequence. Alignments and genome coverage were

respectively visualized and estimated by MapView and Tablet (Milne et al., 2010).

GPGV genome sequence was initially assembled on the available GINV genomic RNA (NC_015220)

by aligning contigs obtained by Velvet. Sequence gaps were filled by sequencing cDNA fragments

amplified with primers designed on the obtained contigs (Supplementary Table 1)cloned with the PCR

cloning kit (Invitrogen, USA). A minimum of four clones per fragment was sequenced. Sequences of

the extreme 3 terminal nts were obtained by rapid amplification of cDNA ends (RACE) using two

independent specific primers (GPgV6643f and GPgV7009f;Supplementary Table 1) and the 5/3

RACE kit from Roche Applied Sciences (Indianapolis, USA).

Phylogenetic analysis was done with Clustal V (Larkin et al., 2007)using 1000 bootstrap replicates

and the neighbor-joining method of analysis.

2.2. Field survey for the detection of GPGV

Forty grapevine plants of the cvs Pinot gris, Traminerand Pinot noirwere identified in different

commercial vineyards originating from the same cultivated area, either showing or not the described

symptomatology. Total RNAs were purified from leaf and petiole tissues by a CTAB-based extraction

protocol according toGambino et al. (2008).GPGV RT-PCR detection was performed by a two-step

cDNA reverse transcription with random hexanucleotide primers and amplification according

toPantaleo et al. (2010)using specific primers (GPgV5619f/GPgV6668r;Supplementary Table 1)

designed on the obtained sequence.

3. Results

3.1. High throughput sequencing and analysis of small RNAs

Libraries representative of the siRNAs population extracted from S1+ and S2 vines and sequenced

by Illumina technology, contained respectively 6.9 106and 1.1 107 reads, after trimming adapters

and filtering for transfer and ribosomal RNAs (Table 1). Only the data relevant for the identification of

virus and viroid-related sequences were analyzed in the present study, whereas a detailed descriptionof the library and of the small interfering RNA (siRNA) species will be the object of another paper.

Table 1. Virus- and viroid-related 1826 siRNAs and contigs originated from both libraries.

Plant S1+ Plant S2

Virus/genus Readsa %c % v and vdd Contigs Readsa %c % v and vdd Contigs

HSVd/Hostuviroid 72,496 1.04 28.15 16 123,019 1.11 23.66 15

GYSVd1/Apscaviroid 63,050 0.90 24.48 6 82,808 0.75 15.92 13

GRSPaV/Foveavirus 9378 0.13 3.64 26 135,996 1.23 26.15 189

GRVFV/Marafivirus 451 0.01 0.18 13,498 0.12 2.60 25

GSyV-1/Marafivirus 368 0.01 0.14 11,800 0.11 2.27 28

GPGV 112,184 1.61 43.56 36 152,924 1.38 29.41 48Total virus reads 257,559 520,045
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Plant S1+ Plant S2

Virus/genus Readsa %c % v and vdd Contigs Readsa %c % v and vdd Contigs

Othere 6,721,320 10,545,472

Total 6,978,879b 11,065,517b

a

Redundant reads obtained by SOAPaligner.

b

Total reads after trimming of the adapters and filtering for ribosomal and transfer RNAs.

c

Percentages over the total reads.

d

Percentages over the total virus and viroidal reads.

e

Reads not matching with viral and viroidal sequences.

Table options

Preliminary BLASTN analysis of sequenced reads against the NCBI viral sequence database,

identified siRNAs originating from the two grapevine viroids GYSVd1 and HSVd, the flexivirus

GRSPaV and the two marafiviruses GRVFV and GSyV-1 (not shown). Libraries were therefore

searched with SOAP using reference genome sequences of the above viruses and viroids.

Vd-siRNAs originating from Hop stunt viroid(HSVd) and Grapevine yellow speckle viroid 1(GYSVd1)

were similarly represented in both plants either relatively to the total siRNAs or to v- and vd-siRNAs

(Table 1). Consistently withNavarro et al. (2009), who reported that viroid RNAs are targeted by

different Dicer enzymes producing 21, 22 and 24 nt siRNAs, we found a comparable size distribution

of vd-siRNAs. In particular, the 21 and 24 nt populations were the most represented molecular

species. However, differently fromNavarro et al. (2009), equal (+) and () HSVd small RNAs

populations were found whereas, in the case of GYSVd1, the () orientation small RNA species

prevailed (Supplementary Fig. 1).

Grapevine rupestris stem pitting-associated virus(GRSPaV) generated a significant amount of v-

siRNAs which, however, considerably differed in number between the two plants, as they represented

3.64% and 26.15% of the total v-siRNAs and vd-siRNAs in vine S1+ and S2, respectively (Table 1).

The size distribution and polarity of GRSPaV-derived siRNAs, that showed the presence of a 21

prevailing v-siRNAs population followed by the 22 mer population both having a major (+) sense

orientation, confirmedPantaleo et al. (2010)claim that the anti-viral component of RNA silencing in

grapevine progresses, for this virus, as observed in other hostvirus combinations (Donaire et al.,

2009)(Supplementary Fig. 2).

Additional siRNAs identified in both PG vines showed homologies to the marafiviruses Grapevine

Syrah virus-1 (GSyV-1) (Al Rwahnih et al., 2009) and Grapevine rupestris vein feathering

virus(GRVFV) (Abou-Ghanem et al., 2003). Consistent with previous results (Pantaleo et al., 2010)
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siRNAs from both marafiviruses showed a prevalence of () sense orientation molecules for both

viruses (Supplementary Figs. 2 and 3).

3.2. De novo assembly of small RNAs

De novo assembly of small RNAs originating from a replicating viral genome during an antiviral

response to infection rests on their overlapping features that allow compiling large continuous

fragments.Kreuze et al. (2009)approach for virus identification was adopted, following assemblage of

1826 short reads into larger contigs by the Velvet specific software. This software generated 4158

and 4837 contigs from vine S1+ and S2, respectively, with sizes ranging between 33 and 470 nts

(S1+) and 33 and 505 nts (S2). Following a search in the NCBI virus database, by BLASTN a

BLASTX, five groups of contigs were identified using a cutoff e-value of 106for BLASTN and 104for

BLASTX (Table 1).

3.3. Re-assembling GRSPaV genomic RNA

The first contigs group showed homology with several isolates of GRSPaV, whose presence in PG

had already been ascertained by short reads analysis and RT-PCR. Vines S1+ and S2 yielded 26

and 189 homologous contigs, respectively, the large majority of which were identified by BLASTN

analysis (Table 1). The almost complete GRSPaV genomic RNA (7731 of the 8725) was manually

assembled from plant S2on the template sequence of genomic RNA of isolate GRSPaV (Zhang et

al., 1998; GenBank ID:AF026278). The choice of this RNA template was determined by the high

number of contigs (149 of 189) showing homologies with the GRSPaV sequence. Of the remaining 40

contigs, 23 had a prevailing homology with GRSPaV-Syrah (GenBank ID:AY368590)throughout the

entire genome, whereas 17 contigs could not be assigned but to a single viral strain. Contigs were

distributed throughout the genomic RNA with the exceptions of 12 nts at the 5terminus and 63 nts at

the 3end, and of a few interspersed regions. All contigs were correctly placed in each open reading

frame (ORF), thus supporting their origin from the de novo assemblage of small RNAs coming from

viable replicating molecules. A full length GRSPaV consensus sequence was obtained by

SOAPaligner, which resulted in a 66 average coverage depth of the genome. This sequence was

registered in the European Bioinformatics Institute (EBI) database under the accession number

HE591388. These findings were interpreted as evidence that the GRSPaV population infecting vine

S2 is made up of a master sequence related to GRSPaV and a minor variant with prevailing

homology to GRSPaV-Syrah.

3.4. Discovery of a novel virus

The second group of contigs, composed of 36 from vine S1+ and 48 from vine S2(Table 1), showed

homology to the 7241 nt genomic RNA of the trichovirusGINV. This finding prompted further

investigations to confirm the presence of GINV-related sequences by alternative methods. RT-PCR

analysis with flexivirid degenerate primers designed in the RdRp replicase domains (Saldarelli et al.,

1999), amplified a 376-nt fragment from both vines, in line with the expectations because of the
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alleged presence in them of the flexivirus GRSPaV. However, one of the five clones from vine S1+

contained a GINV-related sequence (BLASTN e-value 7e81), whereas the remaining four clones and

the five clones from vine S2matched GRSPaV genomic RNA.

The 84 contigs from both vines were mapped on the GINV genomic RNA for a total of 5744 nts, with

the exception of the extreme 5and 3 termini and a few internal gaps. These sequence gaps were

covered by Sanger sequencing of cloned amplicons, obtained by RT-PCR with primers designed on

selected contigs (Supplementary Table 1). These primers amplified cDNAs ranging from 941 to

1904 nt in size, from almost the entire genomic RNA length (Fig. 2). The 3untranslated sequence was

obtained by RACE RT-PCR using the terminal-designed primers GPgV6643f and GPgV7009f

(Supplementary Table 1), whereas repeated attempts to reach the 5 terminal region with the same

technique failed. An RNA consensus sequence was finally assembled by SOAP which resulted in a

molecule 7258 nt in size, excluding the polyA tail which, as specified below, belongs to the apparently

undescribed GINV-related virus, provisionally denoted GPGV. This nucleotide sequence was

registered in the EBI database under the accession number FR877530. SOAP alignments of both S1+

and S2 libraries against the new genome identified 112,184 and 152,924 GPGV v-siRNA,

respectively from plant S1+ and S2 (Table 1), represented by a majority of 21 and 22 nucleotides

size length and an equal distribution of (+) and () sense orientation (Supplementary Fig. 3). Although

GPGV v-siRNAs were fewer in the symptomatic S1+ plant, their percentage, relatively to the total v-

and vd-siRNAs is (43.56%) higher than in the symptomless S2plant (29.41%).

Fig. 2. Coding information of Grapevine Pinot Gris virusgenomic RNA. Open reading frames coding for RNA

dependent RNA polymerase (RdRp), movement protein (MP) and coat proteins (CP) are showed. Thin and thick bars

below the figure indicate respectively, positions of contigs and cDNA amplified regions. Polyadenylated cDNAs

obtained by 3RACE are indicated.

Figure options

GPGV genomic RNA, like that of members of the genus Trichovirus(Adams et al., 2004), consists of

three overlapping open reading frames (ORFs) (Fig. 2). ORF1 is 1865 amino acid (aa) in size

(214 kDa) and encodes, in the order, the replicase-associated proteins, methyltransferase (aa 44

333), helicase (aa 10401277) and RdRp (aa 14471797). It also contains the AlkB domain (aa 730

830), a partial HxD motif, plus all the other motifs and residues essential for Fe 2+coordination (Bratlie

and Drabls, 2005). This protein starts with an AUG codon after a 5 untranslated region of 94

nucleotides. ORF2 codes for a 376 aa polypeptide 42 kDa in size showing homologies with the

movement proteins of GINV andApple chlorotic leaf spot virus(ACLSV) (Table 2). ORF3 encodes the

195 aa (22 kDa) putative coat protein (CP). A 3 untranslated region of 82 nucleotides terminates the

genomic RNA before the polyA tail, whose addition is likely signaled by a AAUAAA sequence at

position 7195. Analysis of these proteins showed strong similarities with the comparable GINV

products with aa identities of 66.1% (ORF1), 64.9% (ORF2) and 71.4% (ORF3) (Table 2). Only the CP

gene had the same length (588 nts) in both viruses whereas replicase and movement proteins are

respectively 5604 nts 1053 nts long in GINV and 5538 nts and 1110 nt long in GPGV. Lower identity

values were found with orthologous ORFs of other species in the family Flexiviridaethat ranged
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between 4 and 37.5% at the aa level (Table 2). These findings were confirmed by the phylogenetic

analysis of RdRp and CP, which clearly grouped GPGV in the genusTrichovirus, in the same clade as

GINV (Fig. 3A and B).

Table 2. Percentage amino acids identities between GPGV encoded proteins and orthologs of species of the

family Flexiviridae.

Genus Virus RdRpol MP CP

Trichovirus GINV 66.1 64.9 71.4

Potexvirus PVX 16.6

Mandarivirus ICRSV 17.9

Allexivirus GarV-A 6.8

Tymovirus TYMV 9.1

Vitivirus GVA 25.2 13 24

Trichovirus ACLSV 37.5 25 28

Capillovirus ASGV 24.8 19 23

Citrivirus CLBV 27.7 12 8

Foveavirus ASPV 24.4 5

Carlavirus PVS 25 5

Unassigned BVX 16.3 4

Unassigned BCV-F 6.9 4

Table options

Fig. 3. Phylogenetic trees constructed with RdRp (A) and CP (B) protein sequences of GPGV and members of the

familyFlexiviridae. In both trees GPGV clusters with GINV in a separate clade. Numbers at branches indicate

occurrence of bootstrap replicates out of the 1000 replicates performed in the test. Bars denote evolutionary distances

corresponding to number of amino acids substitution per site. GenBank ID of the amino acid sequences are:

TYMVAAB92649; ASGVAAP80757; GarV-ANC_003375; GarV-CNC_003376; BVXNC_005132; PVXAAA47167;

PapMVNC_001748; NCLVNC_008266; PVMAAP76207; PVSNC_007289; CNRMVNC_002468;

ASPVNC_003462; BVFNC_002604;ACLSVCAA68080; APCLSVNC_006946; ChMLVNC_002500;

PcMVNC_011552;GVBNC_003602;GVANC_003604;LSVNC_005138;GRSPaVAF026278;CGRMVNC_001946;

ICRSVNC_003093;ShVXNC_003557;ClYMVNC_001753;CLBVNC_003877;GINVNC_015220.

Figure options

3.5. Contigs related to marafiviruses and viroids

BLAST search identified two other groups of contigs showing homologies to GRVFV and GSyV-1 only

from PG vine S2, which contained the highest number of specific reads (Table 1). However, due to

the limited numbers of v-siRNAs, it was not possible to obtain a significant coverage of the genomic

RNAs of the two viruses. These contigs covered 2743 out of 6617 and 1925 out of 6506 nucleotides of

the GRVFV and GSyV-1 genomic RNAs, respectively, and were scattered along the entire genomes.

Contigs homologous to sequences of GYSVd1 and HSVd composed the fifth group. Their analysis

suggests that while the major HSVd variant is the same in the viroid populations of both plants with a

prevalence of contigs homologous to isolate 09-2009-2140 hs (GenBank ID: HQ447057 (9 out of 16

and 9 out of 15 contigs, in vine S1+ and S2, respectively), this is not true for GYSVd1, which

occurred as a prevalent variant 09-2009-2140g1 (GenBank ID: HQ447058) in vine S2 (9 out of 13

contigs) and as a mixture of variants in vine S1+. HSVd and GYSVd1 consensus sequences
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