Distinct isolates of Grapevine rupestris vein feathering virus detected in Vitis vinifera in New Zealand
Grapevine (Vitis sp.) is host to more than 65 viruses (Martelli, 2014d) including grapevine rupestris vein feathering virus (GRVFV); from the genus Marafivirus, family Tymoviridae. As part of a virus investigation in New Zealand, GRVFV was detected from three samples of Vitis vinifera. RNA was extracted using Spectrum Plant Total RNA Kit (Sigma) and treated with deoxyribonuclease RQ1. A cDNA library was constructed and sequenced by High throughput sequencing (HTS) by a paired-end method (2×125 bp) at the Australian Genome Research Facility on an Illumina HiSeq sequencer. The sequences were assembled with Trinity (Grabherr et al., 2011) in the Galaxy web-based platform. The first sample was a Chardonnay clone 8021 collected in November 2016 in the New Zealand Winegrower’s germplasm collection, Lincoln, South Island, New Zealand. From the de novo assembly of the total sequence (93M) one contig (isolate NZ Ch8021; accession no. MF000325) of 6708 nt comprising 1533 reads (average coverage of 28.5 fold) representing a near full length GRVFV genome was detected. This contig shared 80% nt identity with GRVFV isolate CHASS (KY513702). From the same HTS run, GRVFV was also detected from two additional grapevines on two different libraries using the same extraction and sequence analysis methods. The first one was obtained from another Chardonnay collected the same day in the same germplasm collection. From the total 92M reads, a contig (isolate ChTK0004; accession no. MF000326) of 6544 nt was assembled from 1416 reads (27 fold average coverage), with the closest sequence from GenBank being GRVFV isolate Mauzac (KY513701, 86% nt identity). The third GRVFV sequence was detected from a lyophilized sample of a Syrah plant showing early reddening symptoms collected in March 2015 from a commercial vineyard (Hawke’s Bay, North Island). From the de novo transcriptome assembly (99 M reads total) a contig (isolate NZ Sy047; accession no. MF000327) of 2696 nt comprised 299 reads, was detected, with a closest match in GenBank to GRVFV isolate CHASS (KY513702, 84% nt identity). When aligned with the GRVFV reference genome (isolate Mauzac KY513701), the contig aligned from nt 2676 to 5371.The presence of the virus was confirmed in the three samples by RT-PCR and Sanger sequencing using the primers GRVFV_6156F and GRVFV_6600R described in Reynard et al. (2017) and the amplicon showed 100% identity to the sequences described above when available (the two Chardonnay samples MF481199 and MF481200). The addition of these three new sequences of GRVFV increased the knowledge of the virus genetic diversity. Between them, the three isolates shared 80.9 – 83.7% nt identity in their 2696 nt common region (93.2-97% aa identity) and the closest genomes available in GenBank shared only 86.8% nts (isolate Mauzac, KY513701) in the common region. The impact of GRVFV on the health of the grapevine was not assessed, as the three plants tested were co-infected with multiple other viruses and viroids. This is the first report of GRVFV sequence from New Zealand.
Plant & Food Research for funding and New Zealand Winegrowers for financial support through the Rod Bonfiglioli Memorial Scholarship.
Identification of a novel vitivirus from grapevines in New Zealand
The genus Vitivirus was named after the host Vitis of its type member, Grapevine virus A. The genus belongs to the family Betaflexiviridae, subfamily Trivirinae. Members of this genus have filamentous virions of about 725-825 nm by 12 nm with a positive sense RNA genome of 7400 to 7600 nt with five Open Reading Frames (ORFs) (Adams et al., 2004). The polyprotein, encoded by ORF1, comprises the domains required for replication, the putative movement protein is encoded by the ORF3, and the coat protein (CP) by ORF4. The ORF5 codes for a putative RNA-binding protein. In the last International Committee on Taxonomy of Viruses (ICTV) master species list (2016 v1.3 available from https://talk.ictvonline.org/) nine viruses are officially members of the genus Vitivirus, namely, grapevine viruses A, B, D, E and F; Actinidia virus A and B; mint virus 2; and Heracleum latent virus (Bem & Murant, 1979, Blouin et al., 2012, Minafra et al., 2017, Tzanetakis et al., 2007). Three recent accessions on GenBank suggest additional species, arracacha virus V (Oliveira et al., 2017), Agave tequilana leaf virus (NCBI accession KY190215), and grapevine virus K (MF072319).
Material and Methods
During a small scale untargeted virus survey, a novel vitivirus was identified from a Vitis vinifera Chardonnay clone 8021 (VID561 – TK06562) sampled in the New Zealand Winegrowers’ germplasm collection, Lincoln, New Zealand. This plant was imported in 1988 from France and entered the germplasm collection which itself was subsequently moved within New Zealand several times. The origin of the virus infection is undetermined as it may have hitchhiked through importation or be a new infection from within New Zealand. The initial virus sequence information was obtained from small RNA (sRNA) sequencing. Leaf samples were collected in January 2016, and sRNA was extracted using the mirVana microRNA isolation kit (Ambion, Thermo Fischer Scientific). The library was prepared and sequenced on an Illumina HiSeq by BGI. The sRNA analysis was performed using the YABI Virus Surveillance and Diagnosis (VSD) toolkit (Barrero et al., 2017, Hunter et al., 2012) and identified 10 contigs (between 239 to 1333 nt in length), which showed homology to accessions of grapevine virus E (GVE) on GenBank, but only at the protein level (BlastX) with values ranging between 41 to 72% aa homology. This vitivirus-like sequence information was used to design RT-PCR primers and Sanger sequencing of RT-PCR products confirmed the presence and the sequence of the virus. Four viruses and two viroids were also detected from the sample by the VSD toolkit (grapevine leafroll-associated virus 2, grapevine leafroll-associated virus 3 (strain NZ2), grapevine rupestris stem pitting virus, grapevine red globe virus, hop stunt viroid and grapevine yellow speckle viroid). The plant was resampled in November 2016, and total RNA was extracted using Spectrum Plant Total RNA Kit (Sigma), treated with deoxyribonuclease RQ1 and submitted to RNASeq (125 bp pair-end) at the Australian Genome Research Facility on a HiSeq 2500 sequencer. Assembly of the data (93 M reads in total) was performed with Trinity (Grabherr et al., 2011) in the Galaxy web-based platform. All the viruses and viroids detected previously were confirmed with the addition of grapevine rupestris vein feathering virus (Blouin & MacDiarmid, 2017a). Following assembly, the two largest contigs with homology to vitiviruses were 5551 and 1943 nt long and they could be assembled with a 20 nt overlap (100% identical) forming a final contig of 7474 nt that matched the Sanger sequences previously obtained. When used as a reference, this 7474 nt sequence was mapped using Bowtie2 (version 2.2.4) (Langmead & Salzberg, 2012) to 1279 reads from the total RNA sequencing (21 x coverage) and 418685 reads from the sRNA sequencing (1176 x coverage). The genome was completed with the 5´ UTR sequence obtained with the SMARTer® RACE 5´/3´ Kit (Clontech Laboratories, Inc. A Takara Bio Company) and the 3´ UTR by RT-PCR using an oligo(dT) anchored reverse primer.
Results and discussion
The complete genome of the virus is 7496 nt long (MF405923) and contains five ORFs coding for proteins containing all the conserved domains expected in a vitivirus when analysed using the conserved domain database (Figure 3.1a) (Marchler-Bauer et al., 2011). The ORF1 (position 65:5176 nt) encodes a polyprotein of 1710 aa that contains four recognised domains (from the N terminus): metyltransferase; helicase; 2OG-Fe(II) oxygenase superfamily (AlkB); and RNA-dependent RNA-Polymerase (RdRp) at its C terminus (Figure 3.1a). The second ORF starts 14 nt after the stop codon of the ORF1 (position 5191-5655 nt) and encodes 154 aa long protein. It has no recognised domains and no known function, as observed for the other vitiviruses (Martelli et al., 1997, Minafra et al., 2017). The third ORF starts 20 nt after the ORF2 (position 5676-6536 nt) and encodes 286 aa long protein with viral movement protein domain. The ORF4 overlaps ORF3 by 89 nt (position 6448:7053 nt) and encodes a 201 aa protein with the recognised Tricho CP domain. ORF5 starts 36 nt after the stop codon of the ORF4 (position 7090:7443 nt) and is the shortest ORF coding for 117 aa with a viral nucleic acid binding domain. The 5´ and 3´ UTRs are 64 and 53 nt long, respectively (excluding the 3´ poly A tail).
Based on the species demarcation criteria proposed by the ICTV, distinct species is considered within the genus Vitivirus if it shares less than 80% aa identity or 72% nt identity for the CP and the RdRp with its closest relative (Adams et al., 2004). This new sequence is clearly below that threshold and should be considered as a representative of a new species in the genus Vitivirus. We propose to name this new virus grapevine virus G (GVG). Assessment of the biological impact of this virus on the plant is challenging due to difficulty to transmit the virus via inoculation with virus particles (Minafra et al., 2017) and because of the presence of coinfecting viruses. However, future research to extend the survey to additional material to estimate the extent of the virus spread may identify a vine infected with only GVG.
We would like to acknowledge The New Zealand Institute for Plant & Food Research Limited for funding, New Zealand Winegrowers for the financial support through the Rod Bonfiglioli Memorial Scholarship, and Plant Biosecurity Cooperative Research Centre for the funding of the small RNA sequencing and analysis (PBCRC2064). We would like to thank our Plant & Food Research colleagues, John Fletcher for the collection of samples, Ben Warren for his assistance in the bioinformatics, and Kieren Arthur, Kar Mun Chooi, and Dan Cohen for the useful comments on the manuscripts. We would like to acknowledge Dr Maher Al Rwahnih for alerting us of the new genome of GVD available on GenBank.
Grapevine virus I, a putative new vitivirus detected in co-infection with grapevine virus G in New Zealand
The use of High Throughput Sequencing (HTS) has had a massive effect on the rate of discovery of previously overlooked obligate parasites. The decrease of the technology cost combined with the uptake of the methodology by more diagnostic laboratories has resulted in an unprecedented level of detection of novel virus genomes (reviewed in Roossinck, 2017). We reported earlier the finding of a new vitivirus named grapevine virus G (GVG) from small RNA (sRNA) sequencing and total RNA sequencing (Blouin et al., 2018a). While characterising GVG, we identified from the same HTS run a second, related vitivirus. In order to maintain the accepted taxonomical consistency we propose to name this new virus grapevine virus I (GVI) and we use this name hereafter.
Material and Methods
A sample of Vitis vinifera cv Chardonnay (VID499 – TK0004) was collected from the New Zealand Winegrowers’ germplasm collection, Lincoln, New Zealand, in November 2016 and total RNA was submitted to RNASeq at the Australian Genome Research Facility on a HiSeq 2500 sequencer after a DNase RQ1 treatment alongside with the sample VID561 – TK06562 in which the virus GVG was described. Using the bioinformatics pipeline previously described (Blouin et al., 2018a). A vitivirus-like sequence of 7439 nt was retrieved from the de novo analysis. In light of this new sequence, we examined the small RNA (sRNA) data obtained previously (Blouin et al., 2018a). from the same plant and we found that the contig was mapped by 217532 reads (579 x coverage). The sequence was confirmed by Sanger sequencing. The genome was completed with the 5´ UTR sequence by using the SMARTer® RACE 5´/3´ Kit (Clontech Laboratories, Inc. A Takara Bio Company) and the 3´ UTR by RTPCR with an oligo(dT) anchored reverse primer. The full genome of 7507 nt, excluding the polyA tail, was deposited in GenBank, as grapevine virus I, under the accession number MF927925. In addition to this virus, the plant was found infected with several viruses including grapevine leafroll-associated virus 3, grapevine virus A, grapevine rupestris stem pitting virus, grapevine rupestris vein feathering virus (MF000326), grapevine redglobe virus, grapevine virus G (MF405924), and the viroids hop stunt viroid and grapevine yellow speckle viroid, and these were confirmed from the sRNA data using the YABI Virus Surveillance and Diagnosis (VSD) toolkit (Barrero et al., 2017, Hunter et al., 2012).
Results and discussion
The genome of GVI is comparable to the description of vitiviruses (Adams et al., 2004, Minafra et al., 2017) with a single positive single-stranded RNA molecule containing five Open Reading Frames (ORFs). The ORF1 encodes a 1696 aa polyprotein (nt position 69-5159) that contains the recognised domains of methyltransferase; helicase; 2OG-Fe(II) oxygenase superfamily (AlkB); and RNA-dependent RNA-Polymerase (RdRp). The closest relative on GenBank is grapevine virus E (GVE, isolate SA94, GU903012) with 65% aa and nt identity. The second ORF (ORF2) overlaps with the ORF1 by 11 nt (nt position 5149-5652) and codes for a 167 aa putative protein with poor homology to known proteins and no recognised domains, as observed in previously characterised viruses from that genus (Minafra et al., 2017). The third ORF (ORF3) starts 32 nt downstream of the ORF2 and codes for a 264 aa protein (nt position 5685-6479) containing a viral movement protein domain. The movement protein of GVE is its closest relative with 63% aa identity (65% nt). The next ORF (ORF4) overlaps with ORF3 by 70 nt and codes for a 199 aa protein (nt position 6409-7008) containing the tricho coat super family domain. This protein shares 65% aa identity to the coat protein of agave tequilina leaf virus (ATLV) (68% nt); 63% aa with GVE (66% nt) and 62% aa with GVG (61% nt). The ORF5 starts 29 nt downstream of the ORF4 and codes for a 121 aa protein (nt position 70387403) with a recognised viral nucleic acid binding protein (NABP). This protein is the most conserved with GVE the closest match on GenBank (72% aa and 70% nt identity). It is interesting to note that the NABP of Grapevine virus B (GVB) is the second closest match with 66% aa identity as none of the other proteins of the vitivirus GVB group with the GVE clade. A phylogenetic analysis was conducted on the replicase and the coat protein genes from a ClustalW aligment (BLOSUM cost matrix with gap opening cost set at 10 and gap extend cost at 0.1) and made with Neighbor-joining method using the Jukes-Cantor genetic distance model. The citrus leaf blotch virus replicase (JN983456) and apple chlorotic leaf spot virus coat protein (CAE52495) were used as outgroups.
1. GENERAL INTRODUCTION
1.1 NEW ZEALAND WINE HISTORY
1.2 A BRIEF HISTORY OF GRAPEVINE VIRUSES IN NEW ZEALAND
1.3 GRAPEVINE VIRUSES
1.6 HTS APPLICATION IN GRAPEVINE VIROLOGY
2.1 A NEW VIRUS DISCOVERED BY IMMUNOCAPTURE OF DOUBLE-STRANDED RNA, A RAPID METHOD FOR VIRUS ENRICHMENT IN METAGENOMIC STUDIES
2.2 IMPROVEMENT OF DOUBLE-STRANDED IMMUNOCAPTURE FOR GRAPEVINE VIRUS ENRICHMENT
3 NEW VIRUSES DETECTED IN NEW ZEALAND
3.1 DISTINCT ISOLATES OF GRAPEVINE RUPESTRIS VEIN FEATHERING VIRUS DETECTED IN VITIS VINIFERA IN NEW ZEALAND
3.2 IDENTIFICATION OF A NOVEL VITIVIRUS FROM GRAPEVINES IN NEW ZEALAND
3.3 GRAPEVINE VIRUS I, A PUTATIVE NEW VITIVIRUS DETECTED IN CO-INFECTION WITH GRAPEVINE VIRUS G IN NEW ZEALAND
4. SNAPSHOTS OF A COUNTRY’S VINEYARD VIROME
4.2 MATERIALS AND METHODS
5. GENERAL DISCUSSION
5.2 HTS: THE TOOL, ITS FAULTS AND ITS ROLES
5.3 ROLE OF HTS IN PLANT VIRUS DETECTION
5.4 NEW VIRUSES DETECTED
5.6 FUTURE DIRECTIONS
GET THE COMPLETE PROJECT