Supplementary Table 1: Total number of reads, after trimming, associated with each dataset analyzed in
this study, as well as the genome length of each virus, the average coverage of the assembled contigs and the
GenBank accession associated with each genome. GVN – grapevine virus N, GVO – grapevine virus O, nt –
nucleotides.http://link.springer.com/journal/7052023-01-06hj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Le Cordeur, Nathan

Pietersen, Gerhard

Read, David Alan

Swanevelder, Dirk

Thompson, Genevieve Dawn

UPSpace at the University of Pretoria

 1 Genomic characterization of grapevine viruses N and O: Novel vitiviruses from South Africa  David A. Read 1,2*, Genevieve D. Thompson 1,3, Nathan Le Cordeur 4, Dirk Swanevelder 1 and Gerhard Pietersen 2 1 Agricultural Research Council (ARC) - Biotechnology Platform, 100 Old Soutpan Road, Onderstepoort, Pretoria, 0110, South Africa 2 Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa 3 Gene Vantage, 34 Monte Carlo Crescent, Kyalami Business Park, Johannesburg, 1684, South Africa 4 Patho Solutions, Olifantskop Road, Wellington, 7655, South Africa  * - Corresponding author Email: david.read@fabi.up.ac.za ORCID  David A. Read 0000-0002-6614-9946  Abstract  A survey was performed on a Vitis cultivar collection in Stellenbosch, South Africa. Metaviromes were generated for each cultivar, using an RNAtag-seq workflow. Analysis of assembled contigs indicated the presence of two putatively novel members of the genus Vitivirus, provisionally named grapevine virus N (GVN) and grapevine virus O (GVO). Comparisons of amino acid identities showed that GVN and GVO are most closely related to grapevine virus G and grapevine virus E respectively. The incidence of these novel viruses within the sampling site was low, with GVO and GVN associated with only five and two cultivars respectively, of the 229 sampled.   Vitis vinifera L. (grapevine) is a significant crop within the South African agricultural sector, with the country being in the top ten for both wine and table grape production [1]. Vegetative propagation and global trade of cultivars has led to grapevine being disproportionately affected by more viruses than any other crop [2]. The exponential increase in the use of metaviromics has accelerated the discovery of grapevine pathogens, with the number of currently known  2 viruses approaching ninety [3]. South Africa has a long history of viticulture and consequently,  a diverse set of viruses circulating within the industry.   Members of the genus Vitivirus (family Betaflexiviridae) typically have genomes of  approximately 7,500 nt, with five open reading frames coding for the replication associated  proteins (RAP), a 22- Da  ei  a cia ed i h ec  a i i , a movement protein  (MP), a coat protein (CP), and a nucleic acid binding protein (NABP) that functions as  suppressor of RNA silencing [4]. Grapevine vitiviruses are common components of viral  populations in South African vineyards [5], with grapevine virus A (GVA), grapevine virus B,  and grapevine virus E (GVE) having been previously reported [6, 7, 8]. In general, single  infections with grapevine vitiviruses are associated with very mild symptoms [9], however  synergistic co-infections with other viruses, result in several severe and economically  important disease phenotypes [10]. GVA and GVB are part of a complex of viruses associated  with rugose wood [11] and in South Africa, are associated with Shiraz decline and corky bark  disease respectively [7, 12]. In recent years, the application of metaviromics approaches have  led to the discoveries of a number of new grapevine vitiviruses, such as grapevine virus F [11],  grapevine virus G (GVG) [13], grapevine virus H [14] and grapevine virus L [15].   In December 2019, a total of 229 samples were collected from the Vitis cultivar collection  a  he Ag ic a  Re ea ch C ci  (ARC) Nie bi  ca , a  de c ibed e i   [16] (GPS co-ordinates: -33.912053, 18.862291). Total RNA was isolated according to White  et al, [17] and RNAtag-seq libraries were prepared as described [18], which were sequenced  using an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, United States; ARC -  Biotechnology Platform, Onderstepoort, Pretoria, South Africa), as paired-ends (2 x 125 nt)  using TruSeq V4 chemistry (Illumina, San Diego, CA, United States).   Datasets were demultiplexed with Je software [19]. Trimming was performed with CLC  Genomics Workbench 9 (Qiagen Bioinformatics, Aarhus, Denmark), using the following parameter values: Minimum read length of 20 nt, quality limit of 0.05 and adapter trimming with Illumina universal (5 -AGATCGGAAGAG-3 ) and RNAtag-seq (5 -TACACGACGCTCTTCCGATCTNNNNNNNNT-3 ) adapters. Assembly of trimmed reads into contigs was done with SPAdes 3.14.0 [20], with the meta option. The cultivars and datasets associated with this study, are listed in Supplementary Table 1. These datasets are associated with NCBI BioProject PRJNA763365. Contigs of putative viral origin, were identified using BLASTn [21] and the viral fraction of the NCBI Refseq database. Plant virus contigs were then submitted to the browser-based version of BLASTx, using the NCBI nr database, for further confirmation of their identity.  3 A novel vitivirus, provisionally named grapevine virus N (GVN), was associated with V. vinifera cultivars Azal tinto, Bourboulenc, Cinsault noir, Crouchen blanc and Palamino, while the provisionally named grapevine virus O (GVO) was associated with Roter Zierfandler and Royal Molenberg. The GVN and GVO genome organizations and associated amino acid sequences of each gene were determined using ORF finder [22], showing the typical arrangements of the genus Vitivirus [11]. The average amino acid identity (AAI) between each gene product and that of the closest relative was determined using the AAI calculator from the enveomics collection [23] and shown in Table 1. The amino acid sequences of ORF1 (RAP) of both GVN and GVO were aligned against cognate NCBI RefSeq sequences of extant vitiviruses, using BioEdit 7.2.5 [24]. A best-fit maximum likelihood phylogeny, based on the Le Gascuel model + G + I + F, was generated using MEGA X [25] and shown in Figure 1.  Partial segments of the GVN and GVO CP gene were amplified using RT-PCR, for each respective sample. Primers with the following sequences were used to set up two-step RT-PCR reactions: GVN-CP-F (5 -TCGCTGAGATAATAAGGAGGATTGAG-3); GVN-CP-R (5 -GACTTGAATCACACTGGCTTCAGA-3 ); GVO-CP-F (5 -GGTGTGATAGAGGAT AACCACAGT-3 ) a d GVO-CP-R (5 -TACACTCTAAACGACCACAACAGT-3 ). Two-step RT-PCR reactions were carried using Promega GoScriptTM Reverse Transcriptase and GoTaq® Taq polymerase (Promega, Madison, WI, United States), according to manufacturer instructions. Amplicons of the expected size were visualized on an agarose gel (572 and 789bp for GVO and GVN respectively) and the identities confirmed using bidirectional Sanger sequencing (Inqaba Biotechnical Industries, Pretoria, South Africa). The 5  terminal nucleotides of GVN and GVO were confirmed using the 5' RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen, Carlsbad, CA, United States) according the to manufacturer s specifications. Total RNA from Palamino and Roter Zierfla de  a  ed a  he i  f  GVN a d GVO 5  RACE eac i  e ec i e . The  following gene specific primers were used for GVN (GVN-GSP1: 5 - CACTATATCTTAACTCATCT-3  and GVN-GSP2: 5 - CCTCTACAATATGACTAGATATGCT-3 ) and for GVO (GVO-GSP1: 5 - AGGGTCGTCTTATCTTCATC-3  and GVO-GSP2: 5 -TCCCTATACCTTAGGTTATC CTTAGC-3 ). 3  RACE amplification was not performed as all of the GVN and GVO contigs were associated with a poly-A-tract between 48 and 52 nt in length (data not shown). C fi a i  f he 5  ends showed that the GVN and GVO genomes are 7,486 and 7,560 nt respectively (excluding poly-A-tracts).   4 Considering the RAP phylogeny, GVN and GVO are most closely related to GVG and GVE respectively, which is also confirmed by AAI shared between the various genes of these viruses. Phylogenetically, GVN and GVO both cluster within the recently ascribed GVE superclade [26]. AAI indicates that GVN and GVO are novel viruses within the Betaflexiviridae family where the species demarcation limit is less than 80% AAI for either the RAP or CP genes [27]. However, some additional criteria may need to be implemented in order to determine whether GVN and GVO are representatives of two new species or distinct members of already established taxa. GVN and GVO showed limited incidence and genetic diversity within the virus population of the vineyard under study, which could suggest a recent i d c i . H e e , e ide ead e  f S h Af ica  i ic a  egi  is needed in order to get a complete view on their distribution nationwide. Finally, the role of GVO and GVN in any grapevine disease requires further investigation, as both viruses were found in co-infections with other viruses including grapevine leafroll-associated virus 3, grapevine leafroll-associated virus 2 and at least two additional grapevine vitiviruses (data not shown). Given the potential for grapevine vitiviruses to be associated with synergistic co-infections [9], GVN and GVO should be considered possibly damaging pathogens for South African viticulture.  Declarations   Funding: David Read is grateful for the financial support provided by the National Research Foundation of South Africa, under grant UID 104901.  Conflict of interest: All authors declare that they have no conflict of interest.  Availability of data and material: The data that support the findings of this study, are openly available in NCBI public databases.  Code availability: Not applicable  Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.    5 References    1 Gbejewoh O, Keesstra S, Blancquaert E (2021) The 3Ps (Profit, Planet, and People) of  Sustainability amidst Climate Change: A South African Grape and Wine Perspective.  Sustainability 13:2910    2 Martelli GP (2018) Where grapevine virology is heading to. In: Proceedings of the 19th  congress of the International Council for the Study of virus and virus-like diseases of the  grapevine (ICVG), Santiago, Chile, 9-12 April 2018    3 Fuchs M (2020) Grapevine viruses: a multitude of diverse species with simple but overall  poorly adopted management solutions in the vineyard. J Plant Pathol 102:643 653    4 Galiakparov N, Tanne E, Sela I, Gafny R (2003) Functional analysis of the grapevine virus  a genome. Virology 306:42 50.    5 du Preez J, Stephan D, Mawassi M, Burger JT (2011) The grapevine-infecting vitiviruses,  with particular reference to grapevine virus A. Arch Virol 156:1495 1503    6 Engelbrecht DJ, Kasdorf GGF (1987) Occurrence and transmission of grapevine virus A in  South African grapevines. South African J Enol Vitic 8(1):23-29.    7 Goszczynski DE (2010) Divergent molecular variants of Grapevine virus B (GVB) from  corky bark (CB)-affected and CB-negative LN33 hybrid grapevines. Virus Genes 41:273 281    8 Goszczynski DE, Jooste AEC (2003) Identification of divergent variants of Grapevine virus  A. Eur J Plant Pathol 109:397 403    9 Rowhani A, Daubert S, Arnold K, Al Rwahnih M, Klaassen V. Golino D, Uyemoto JK (2018)  Synergy between grapevine vitiviruses and grapevine leafroll viruses. Eur J Plant Pathol 151: 919 925    6 10 Rowhani A, Uyemoto JK, Golino D, Daubert SD, Al Rwahnih M (2016) Viruses involved in graft- incompatibility and decline. In Meng B, Fuchs M, Martelli G Golino D (eds) Grapevine viruses: Molecular biology, diagnostics, and management, Chapter 12. Springer, New York, pp 289 302  11 Al Rwahnih M, Sudarshana MR, Uyemoto JK, Rowhani A (2012) Complete genome sequence of a novel vitivirus isolated from grapevine. J Virol 86(17):9545  12 Goszczynski DE, du Preez J, Burger JT (2008) Molecular divergence of Grapevine virus A (GVA) variants associated with Shiraz disease in South Africa. 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Biotechnol J 3:1424 1428  18 Shishkin AA, Giannoukos G, Kucukural A et al (2015) Simultaneous generation of many RNA-seq libraries in a single reaction. Nat Meth 12:323 325  19 Girardot C, Scholtalbers J, Sauer S et al (2016) Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers. BMC Bioinformatics 17(1):419  7  20 Nurk S, Meleshko D, Korobeynikov A, Pevzner PA (2017) metaSPAdes: a new versatile metagenomic assembler. Genome Res 27(5):824-834  21 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403 410  22 Wheeler DL, Church DM, Federhen S, Lash AE, Madden TL, Pontius JU, Schuler GD, Schriml LM, Sequeira E, Tatusova TA, Wagner L (2003) Database resources of the National Center for Biotechnology. Nucleic Acids Res 31:28-33  23 Rodriguez-R LM, Konstantinidis KT (2016) The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. 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Length (nt) Gene Gene location (nt) Product size (aa) Putative gene function/s MW (kDa) pI AAI GVN 25-01 Palomino/ MZ682355 7,486 RAP 64-5178 1704 Replication 194 5.9 70.61 ORF2 5184-5660 158 Hypothetical protein 17.7 6.5 36.81 MP 5681-6529 282 Movement protein 31.6 5.8 67.31 CP 6441-7046 201 Coat protein 21.8 7.8 80.11 NABP 7081-7413 110 RNA-binding protein 12.6 7.4 87.01 GVO 28-12 Roter Zierfandler/ MZ682356  7,560 RAP 68-5161 1697 Replication 192 6.1 75.62 ORF2 5158-5724 188 Hypothetical protein 21.2 6.8 53.32 MP 5752-6558 268 Movement protein 29.6 6.0 77.72 CP 6434-7078 214 Coat protein 23.6 9.0 87.42 NABP 7096-7425 109 RNA-binding protein 12.6 9.6 69.12 Table 1: Data on grapevine virus N (GVN) and grapevine virus O (GVO) genomes and deduced genome products. AAI - amino acid sequence identity shared between the viruses in this study and cognate gene products the most closely related viruses MW  molecular weight in kilodaltons; pI  isoelectric point;  RAP  replicase-associated proteins;  ORF2  open reading frame 2; MP  movement protein; CP  coat protein; NABP  nucleic acid binding protein.  1 NC040616 grapevine virus G 2 NC011106 grapevine virus E  9                                 Figure 1: Maximum likelihood phylogeny based on the amino acid sequences derived from ORF 1 (replicase associated proteins; RAP) of grapevine virus N and grapevine virus O (indicated by solid circle markers) from this study and references derived from other extant vitiviruses. The cognate RAP sequence from potato virus T was used as an outgroup. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the Le Gascuel model with frequencies, invariant sites and gamma distribution (n=5). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown.   

Genomic characterization of grapevine viruses N and O : novel vitiviruses from South Africa

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