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Virology Journal is an open access, peer reviewed journal that considers articles on all aspects of virology, including research on the viruses of animals, plants and microbes. The journal welcomes basic research as well as pre-clinical and clinical studies of novel diagnostic tools, vaccines and anti-viral therapies.

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Clinical Virology : Fred Kibenge, University of Prince Edward Island, Canada  Emerging viruses : Tom Geisbert,  University of Texas Medical Branch, USA Hepatitis viruses : Wan-Long Chuang,  Kaohsiung Medical University, Taiwan Herpes viruses : Tony Cunningham,  The Westmead Institute for Medical Research, Australia Influenza viruses : Hualan Chen,  Chinese Academy of Agricultural Sciences, China Negative-strand RNA viruses : John Barr,  University of Leeds, UK Other viruses : Erna Geessien Kroon,  Universidade Federal de Minas Gerais, Brazil Plant viruses : Supriya Chakraborty,  Jawaharlal Nehru University, India Positive-strand RNA viruses : Jaquelline Germano de Oliveira,  Fundação Oswaldo Cruz - Fiocruz, Brazil  Public health :  Kin On Kwok,  The Chinese University of Hong Kong Retroviruses : Aguinaldo Pinto, Universidade Federal de Santa Catarina, Brazil Veterinary DNA viruses : Walid Azab,  Freie Universität Berlin, Germany Veterinary RNA viruses : James Weger-Lucarelli,  Virginia Tech, USA Viruses of microbes : Joana Azeredo,  University of Minho, Portugal

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Alan McLachlan, Co-Editor-in-Chief

Alan McLachlan is a molecular geneticist and hepadnavirologist. He currently serves as a Professor in the Department of Microbiology and Immunology, University of Illinois Chicago, USA. His interests are focused on hepatitis viruses, primarily on hepatitis B virus (HBV) and its relationships to liver physiology.  His research is currently directed toward understanding the relationships between HBV transcription and viral biosynthesis using both cell culture and animal models.  His long-term goals include the identification of cellular gene products as targets for the development of small molecular weight antiviral compounds which, in combination with current nuceot(s)ide analog therapeutics, will resolve chronic HBV infections.

Leo Poon, Co-Editor-in-Chief

Leo Poon is a molecular virologist. He currently serves as a Professor in the School of Public Health, The University of Hong Kong and as a co-director of HKU-Pasteur Research Pole. He has strong interests in emerging viruses, including coronavirus and influenza virus. He researches on different aspects of these viruses, ranging from basic virology to clinical diagnosis. His ultimate goal is to use scientific findings to inform public health policy. Over the years, he has published about 290 peer-reviewed articles. Thus far, his work has been cited over 41,000 times and he has an H-index of 95 (Web of Science).

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Virology is the study of viruses and virus-like agents, including, but not limited to, their taxonomy, disease-producing properties, cultivation and genetics.

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Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches

Anja pecman.

1 Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia

2 Jožef Stefan International Postgraduate School, Ljubljana, Slovenia

Denis Kutnjak

Ion gutiérrez-aguirre.

3 Fera Science Ltd., York, United Kingdom

Neil Boonham

4 Institute for Agri-Food Research and Innovation, Newcastle University, Newcastle upon Tyne, United Kingdom

Maja Ravnikar

Associated data.

Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus , discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).

Introduction

Plant viruses and viroids are important plant pathogens, causing economic losses by reducing crop quality and quantity all over the world (Loebenstein, 2008 ; Soliman et al., 2012 ). Thus, their reliable detection is of a crucial importance for plant protection. Classical methods in plant virus diagnostics can be roughly divided into specific (serological/molecular tests) and non-specific (indicator test plants, electron microscopy) approaches. Specific methods are usually targeted to one or a few viral species and require a priori knowledge of the pathogens being tested, whilst non-specific approaches do not require specific knowledge of the pathogens, however, frequently only classify viruses at a genus level based on the shared physical/biological characters. Discovery of new viruses/viroids and new hosts has increased rapidly after the introduction of next generation sequencing (NGS). NGS technologies allow a generic approach (non-specific method) to virus identification that does not require any prior knowledge on the targeted pathogens but can deliver a species/strain specific result (Adams and Fox, 2016 ). It was first employed for plant virus detection in 2009 (Adams et al., 2009 ; Al Rwahnih et al., 2009 ; Kreuze et al., 2009 ). Since 2009, different sample preparation methods have been developed, relying on different nucleic acid inputs, most commonly: total RNA (totRNA); ribosomal RNA depleted total RNA (rRNA depleted totRNA); double stranded RNA (dsRNA); virus derived small interfering RNA (sRNA); RNA from purified or partially purified viral particles; polyadenylated RNA (poly(A) RNA); and RNA after subtractive hybridization with healthy plant RNA. Applications of different sample preparation methods are reviewed in Roossinck et al. ( 2015 ); Wu et al. ( 2015 ), and Adams and Fox ( 2016 ). Viruses have diverse genome organizations and use different replication strategies. Based on these two characteristics they can be classified into 7 groups (the Baltimore classification): double stranded DNA (Group I, dsDNA +/−), single stranded DNA (Group II, ssDNA +), double stranded RNA (Group III, dsRNA +/−), positive sense single stranded RNA (Group IV, ssRNA +), negative sense single stranded RNA (Group V, ssRNA −) viruses, positive sense single stranded RNA viruses that replicate through a DNA intermediate (Group VI, ssRNA-RT +), and double stranded DNA viruses that replicate through a RNA intermediate (Group VII, dsDNA-RT +/−) (Baltimore, 1971 ). Viroids are classified into two families: members of Avsunviroidae family replicate in chloroplast, whereas members of Pospiviroidae family replicate in nucleus (Flores et al., 2014 ). Considering the diversity of viruses and viroids, with different genome organizations in mind, it is conceivable that using different nucleic acid inputs for NGS could affect their overall detection.

Sample preparation methods (i.e., different nucleic acid inputs), used before NGS, can differ in their efficiency and can have specific advantages and disadvantages. For example, subtractive hybridization of the host plant nucleic acids, using tomato ( Solanum lycopersicum ) and Pepino mosaic virus (PepMV, RNA +, Potexvirus, Alphaflexiviridae ) as a model system, resulted in three times more PepMV sequences in subtracted sample (Adams et al., 2009 ), but as it is a time consuming procedure, which requires a healthy plant of the same species as the sample to be tested (Adams and Fox, 2016 ), subtractive hybridization is not well suited in a high-throughput diagnostic settings. Some sample preparation methods may cause bias in the detection of a particular group of viruses. Sequencing of dsRNA was mainly used for detection of RNA + and RNA +/− viruses, since RNA—and DNA viruses could be missed (Roossinck et al., 2015 ) using this approach; nevertheless, a new geminivirus (DNA +) was identified using dsRNA sequencing (Al Rwahnih et al., 2013 ). RNA isolated from purified viral particles has been successfully used for sequencing different viruses (reviewed in Roossinck et al., 2015 ; Wu et al., 2015 ). A comparison between deep sequencing of sRNAs and RNA isolated from viral particles showed higher efficiency of the latter for the reconstruction of complete consensus Potato virus Y (RNA +, Potyvirus, Potyviridae ) genomes (Kutnjak et al., 2015 ). However, virus purification is not applicable for un-encapsidated viruses and requires sample specific processing since it is unlikely that all viruses could be captured by a single protocol for viral particles purification (Roossinck et al., 2015 ; Wu et al., 2015 ). Poly(A) RNA based enrichment strategy has been also used for both RNA and DNA viruses but it is not applicable for the detection of viruses without a poly(A) tail (Wu et al., 2015 ). Data from sequencing poly(A) RNA showed a lower degree of virus genome coverage in comparison to saturated genome coverage reached with sRNA data for Grapevine leafroll-associated virus 3 (RNA +, Ampelovirus, Closteroviridae ), yet a comparison between poly(a)RNA and sRNA data for Hop stunt viroid , ( Pospiviroidae family) showed comparable outcomes (high genome coverage) for both approaches (Visser et al., 2016 ).

In this study, we focused the comparison (with the detection and identification of plant viruses and viroids in mind) on the two types of RNA inputs: sequencing of sRNA and sequencing of rRNA depleted totRNA. Those two approaches seem to be the most generically applicable to viruses with different genome types and replication strategies and could be relatively easily integrated in workflows of diagnostic labs.

Sequencing and assembly of viral sRNA (Kreuze et al., 2009 ) has been successfully used for detection and identification of several plant viruses and viroids and their complete genome assembly (reviewed in Boonham et al., 2014 ; Kreuze, 2014 ). It has been speculated that this approach could be problematic if used to detect viruses that either do not trigger silencing responses or that express silencing suppressors (Roossinck et al., 2015 ). Also, de novo assembly of longer viral contigs could be complicated due to short reads lengths (Boonham et al., 2014 ; Roossinck et al., 2015 ; Adams and Fox, 2016 ). On the other hand, the approach is very generic, using the same protocol of sample preparation for many different plant species and doesn't require high quality of RNA input (Kutnjak et al., 2017 ).

Sequencing of plant viruses using total RNA as an input was first described by Adams et al. ( 2009 ) and Al Rwahnih et al. ( 2009 ), followed by several successful studies (reviewed in Boonham et al., 2014 ). It is also a very generic approach, however, a potential shortcoming of that method can be the low viral RNA titer within the background plant RNA. To overcome this, removal of the highly abundant plant ribosomal RNA from the total RNA pool (rRNA depleted tot RNA) has been explored, which can results in a 10-fold enrichment of viral RNA (Adams and Fox, 2016 ).

Recent comparison (Visser et al., 2016 ) of sRNA and rRNA depleted totRNA for Citrus tristeza virus (RNA +, Closterovirus, Closteroviridae ) and Citrus dwarfing viroid ( Pospiviroidae family) implied a preferential use of rRNA depleted totRNA for de novo assembly of viral genome sequences from NGS data. No wider comparison of these two approaches (including viruses with different genome characteristics) has been reported. With this in mind, our aim was to compare the two approaches, including plant viruses with different genome structures and replication strategies (belonging to different Baltimore classification groups) and viroids from different families into comparison. The aims were to compare the two approaches in terms of: (1) known virus detection and identification (2) recovery of virus/viroid reads and (3) effectiveness of detection of new/unknown viruses by reconstruction of longer viral contigs by de novo assembly and read mapping analysis approaches.

Materials and methods

Description of samples.

Nine virus-infected plant samples were included in this study. The selection included samples of different plant species, infected with a range of plant viruses in single or mixed infections with at least one representative from each group of the Baltimore viral classification containing plant viruses, and viroids from both families (Table ​ (Table1 1 ).

Samples included in the comparison with corresponding results from: NGS (viruses/viroids listed in the table were detected in corresponding samples by NGS) and other diagnostic methods (ELISA, RT-PCR and RT-qPCR).

Taxonomic classification, Baltimore classification and genome organization of detected viruses are given in separate columns. Host plant information is given in the separate column. NA, not applicable; +, detected; –, not detected;

Sample preparation and sequencing

Total RNA was isolated from plant samples using TRIzol reagent (Life technologies, USA) following the manufacturer's instructions. Isolated total RNA was then divided in half for comparative purposes. One half was sent to Seqmatic LLC (USA) for sRNA library preparation (TailorMix miRNA Sample Preparation Kit V2, SeqMatic LLC, USA) and sequencing. The samples were multiplexed in one lane of a HiSeq 2500 (Illumina, USA) in 1 × 50 bp mode. The remaining total RNA was further purified using an RNeasy protocol including DNase treatment following the manufacturer's protocols (RNA Cleanup protocol; RNeasy Mini Kit; Qiagen, Netherlands). Ribosomal RNA was depleted from the purified total RNA and sequencing libraries were prepared using the ScriptSeq™ Complete Kit (plant leaf) (Illumina, USA). The libraries were sequenced using MiSeq (Illumina, USA) in 2 × 300 bp (V3) mode. Number and average length of sequencing reads for every sample sequenced by both approaches are in Supplementary Table 7 .

Detection of viruses in NGS data

Reads obtained by both sequencing procedures were trimmed, filtered and further analyzed to confirm the presence of viruses and viroids. Bioinformatics pipelines used for virus detection from NGS data are detaily described in Supplementary Data 1.1 . In both cases, the presence of suspected viral sequences was confirmed by mapping the reads to the complete viral genome sequences of the most similar viral isolates from the NCBI GenBank database, followed by visual inspection of individual mappings.

Confirmatory testing

The presence of virus in each case was also confirmed by using ELISA, RT-PCR, and RT-qPCR methods (Table ​ (Table1). 1 ). ELISA was performed using polystyrene microtiter plates (nunc-Immuno™, Sigma-Aldrich Inc., USA) and kits containing virus specific reagents as follows, AMV: Cat No. 07001S (Loewe Biochemica GmbH, Germany), CaMV: Cat No. 07086 (Loewe Biochemica GmbH, Germany), PVY: Cat No. 1105 (Bioreba AG, Switzerland) and TYLCV: Cat. No. 1072 (Neogen Europe Ltd., UK). The assays were performed following the manufacturer's instructions. In each case a negative control corresponding to the same species as the test sample was used. The result was considered positive when the optical density (OD) A 405 value after 2 h for a given sample was greater than 2 × the mean OD value of the corresponding negative control. For reverse transcription quantitative PCR (RT-qPCR) and reverse transcription conventional PCR (RT-PCR), total RNA was extracted from fresh or lyophilized plant material using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer instructions. RT-qPCR was performed using published methods for PepMV (Gutiérrez-Aguirre et al., 2009 ) and for ToMV (Boben et al., 2007 ). Conventional RT-PCR was performed for PNYDV (Gaafar and Ziebell, 2016 ), STV (Sabanadzovic et al., 2009 ), ToCV (Dovas et al., 2002 ), TMV (Kumar et al., 2011 ), PLMVd (Loreti et al., 1999 ) TASVd and CLVd (Verhoeven et al., 2004 ). PCR primers designed specifically to confirm the presence of novel CCyV1 were as follows: CCyV1-fw (5′-GTCTCTCTTGCGTTGAGCCA-3′) and CCyV1-rev (5′-GGTTGCGGATAGCTCTTCCT-3′). All the amplicons obtained by RT-PCR were purified and sent for Sanger sequencing (GATC Biotech AG, Germany). The Sanger sequences were aligned against the genomes of detected viral species and their identity was confirmed in all of the cases.

Construction of consensus viral/viroid genome sequences

For every identified virus/viroid the consensus viral genomes were extracted from the sRNA read mappings (see section Detection of Viruses in NGS Data) to obtain a corrected consensus genome. Validation of each corrected consensus genome was performed by mapping the de-novo generated contigs obtained by both NGS approaches to corresponding corrected consensus genome. Both mapping results were visually inspected for possible differences between the de-novo contigs and corrected consensus genome sequence. Observed conflicts were further investigated by inspecting the read mapping results. Finally, few of the observed differences were explained as polymorphisms in viral populations. In sample III, two divergent strains (80% nucleotide identity) of PepMV were detected (PepMV-EU and PepMV-CH2). In this case, the complete genome sequences of the two most similar isolates from NCBI GenBank were used in subsequent comparisons ( {"type":"entrez-nucleotide","attrs":{"text":"KF718832.1","term_id":"681870045","term_text":"KF718832.1"}} KF718832.1 and {"type":"entrez-nucleotide","attrs":{"text":"JX866666.1","term_id":"449042838","term_text":"JX866666.1"}} JX866666.1 ), without the corrections after reads and contigs mapping as described previously.

Comparison of sRNA and rRNA depleted totRNA inputs

For comparisons, all raw reads were trimmed and filtered in CLC Genomic Workbench 9 (Qiagen). For rRNA depleted totRNA datasets, reads shorter than 100 nucleotides were discarded. Then, reads were trimmed using quality scores, setting the limit to 0.05 (see CLC Genomics Workbench User Manual, Chapter 23, for explanation). For sRNA reads, first, adaptor trimming was performed, then reads shorter than 20 and longer than 24 nucleotides were discarded.

First, the viral fraction of the total nucleotides sequenced (from now on called percentage of virus/viroid nucleotides) in each of the datasets for each of the detected viruses was calculated by mapping the trimmed and filtered reads (of the corresponding dataset) to the consensus viral/viroid genomes generated in the previous step. Mapping parameters are listed in Supplementary Tables 1 , 4 .

To further compare the effectiveness of both approaches for detection and discovery of selected viruses, we then performed a normalization by subsampling the data from each sample (for both sRNA and rRNA depleted totRNA) to the same number of nucleotides. Random subsampling was performed to different subsample sizes: 1, 10, 30, and 50 million nucleotides. This was repeated ten times for each sample/size combination, yielding in total 360 datasets (9 samples × 4 subsample sizes × 10 replicates of subsampling). For those, the following analyses were implemented: (1) reads were mapped to the corresponding consensus viral/viroid genomes and the fraction of viral/viroid genome covered by reads (from now on: genome coverage (reads)) and the average depth of sequencing (number of times a nucleotide in a reference is covered by reads averaged for the complete genome) were calculated; (2) de novo assembly of reads was performed using CLC Genomics Workbench 9, followed by mapping the resulting contigs to the corresponding consensus viral/viroid genomes and calculation of the fraction of viral/viroid genome covered by the de-novo contigs (from now on: genome coverage (contigs)). Results of these comparisons are jointly shown in Figure ​ Figure2 2 and visualized as dots connected with solid lane (representing rRNA depleted totRNA results) and triangles connected with dashed lines (representing sRNA results). The mapping and de novo assembly parameters are listed in Supplementary Tables 1 – 4 .

Comparison of sRNA and rRNA depleted totRNA approaches using data size-normalized subsamples. Results for each virus included in the analysis are shown along the x-axis and are grouped according to Baltimore classification (A) Fraction (%) of virus nucleotides in trimmed and filtered complete NGS datasets. (B) Average depth (number of reads covering a position in a viral genome, averaged over the complete genome sequence) at different subsample sizes. Symbol ~ indicate interruption of log scale, below, 0 values are plotted. (C) Fraction of viral genome (in %) covered by reads [genome coverage (reads)] at different subsample sizes. (D) Fraction of viral genome (in %) covered by contigs [genome coverage (contigs)] at different subsample sizes. For (B–D) Dots/triangles represent the mean, whereas vertical bars connect minimum and maximum results of 10 repeated analyses. Four different subsample sizes were used (1, 10, 30, and 50 million nts) and are designated in the first column, other columns follow the same logic. Triangles and dashed lines represent results for sRNA approach, dots and solid lines represent results for rRNA depleted totRNA. In some cases data points are missing, since the size of the complete dataset was smaller than the largest subsample.

Sample characterization

Twelve different viruses (among those, one viral species with two divergent strains) and three viroid species were detected using NGS in the nine samples included in the analysis (Table ​ (Table1). 1 ). Nine were known to be present in the samples before the NGS analysis (marked with * in Table ​ Table1), 1 ), whilst six virus/viroid species were detected using NGS during the study and their presence was confirmed as described in section Materials and Methods (Table ​ (Table1). 1 ). Both methods revealed the presence of 14 viral/viroid species whilst 1 virus (a putative novel viral species from the genus Cytorhabdovirus : CCyV1) could only be detected using the rRNA depleted totRNA approach. Seven samples (I, IV-IX) contained single viral/viroid infections, one sample (II) was infected with two viruses. Sample III was infected with five viruses and one viroid. All of the viruses and viroids detected and included in the study are listed in the Table ​ Table1 1 .

Percentage of virus/viroid reads differs for different viruses

First, we estimated what percentage of the total sequenced nucleotides were viral/viroid nucleotides (of the complete cleaned NGS datasets) for different viral species for each of the two approaches. The percentage of viral/viroid nucleotides was in some cases higher using sRNA input and in other cases higher using rRNA depleted totRNA input (Figure ​ (Figure1). 1 ). Specifically, the results showed that for 6 viruses/viroids the sRNA approach generated a higher fraction of viral/viroid sequences: TASVd, ToCV, CLVd, TYLCV, PNYDV, PLMVd, and PVY (Figure ​ (Figure1: 1 : the viruses located below the diagonal line). For the sRNA approach, the highest percentage of viral sequences was observed for PVY (50%, Figure ​ Figure2A). 2A ). The rRNA depleted totRNA approach generated more viral sequences for 6 viruses: a novel Cytorhabdovirus , PepMV (two isolates), CaMV, AMV, CSNV and TMV (Figure ​ (Figure1, 1 , the viruses located above the diagonal line), with the highest viral sequences fractions for TMV (83%), AMV (56%), CSNV (48%), and CaMV (48%) (Figures ​ (Figures1, 1 , ​ ,2A). 2A ). In two cases (STV and ToMV), the percentage of virus sequences were extremely low regardless of the RNA inputs (Figures ​ (Figures1, 1 , ​ ,2A 2A ).

Fraction of virus/viroid nucleotides (nt) in NGS datasets for small RNA and rRNA depleted totRNA approaches. (A) The plots show the percentage of nucleotides (fraction of total) that mapped to the corresponding consensus viral/viroid genome for both sRNA (x-axis) and rRNA depleted totRNA (y-axis) inputs. Dots represent the value for each virus/viroid (also listed in Figure ​ Figure2A), 2A ), with viral/viroid species abbreviations (see Table ​ Table1) 1 ) next to the dots. The diagonal solid line represents theoretical scenario in which percentage of virus/viroid nucleotides would be equivalent for both approaches; dots above the line represent cases for which fraction of virus/viroid nucleotides was higher using rRNA depleted totRNA input, dots below the line represent cases for which fraction of viruses/viroids nucleotides was higher using sRNA input. (B) Enlarged part of (A) (0–15%).

Comparison on normalized subsamples

To be able to compare the two approaches in a greater detail, we subsampled all of the datasets to the same number of nucleotides. Ten replicates of four different sizes of subsamples (1, 10, 30, and 50 million nucleotides) were generated for each dataset to enable an assessment of the impact of data rarefaction and data variability on the performance of tested parameters.

First, average depth was evaluated (Figure ​ (Figure2B). 2B ). In all cases, average depth increased with the increase of subsample sizes and followed the patterns observed when comparing the fractions of viral sequences nucleotides recovered by the two approaches. Results from 10 independent replicates for each subsample size showed a low variability for PVY, ToCV, PepMV, AMV, TMV, CSNV, and CaMV. Variability between the subsamples in average depth was higher for all other viruses/viroids (Supplementary Table 5 ).

Secondly, we investigated how effectively the reads cover the genomes of different viruses by calculating the fraction of the genome covered by reads [genome coverage (reads)] (Figure ​ (Figure2C). 2C ). Results of the analysis showed low variability between replicates of subsamples, except when mapping rRNA depleted totRNA reads to ToMV, STV, TYLCV, TASVd, and PLMVd where variation was very high (Supplementary Table 5 , Figure ​ Figure2C). 2C ). In all cases, as expected, better genome coverage was achieved with the increasing subsample sizes. For the sRNA approach, complete genomes (100%) were covered for majority of the viruses/viroids at subsample size of 30 million nucleotides. The exceptions were ToMV, STV and the putative novel Cytorhabdovirus . For those, even at 50 million nucleotides, genome coverage was 70% or less.

For the rRNA depleted totRNA approach, for half of the viruses (PVY, PepMV, AMV, TMV, novel CCyV1, CSNV, CaMV, CLVd, and TASVd) complete genomes were covered at 10 million nucleotides. However, for some viruses/viroids (ToCV, TYLCV, PNYDV, and PLMVd) relatively low genome coverage was achieved at smaller subsample sizes (1 and 10 million nts) and even at the largest subsample size (50 million nts) the coverage did not reach 100% (Figure ​ (Figure2C). 2C ). The genomes of ToMV and STV, for which very low numbers of reads were recovered (Figures ​ (Figures1, 1 , ​ ,2A), 2A ), were poorly covered even at high subsampling depths, for example, even with 50 million nucleotides, coverage remained below 50% (Figure ​ (Figure2C 2C ).

Reads from normalized datasets were de novo assembled into contigs, which were then mapped to the corresponding consensus viral genomes in order to calculate the fraction of the viral genomes covered by the de novo assembled contigs [genome coverage (contigs)] (Figure ​ (Figure2D). 2D ). The analysis of subsample replicates showed in general lower variability for sRNA datasets than rRNA depleted totRNA datasets (Supplementary Table 5 ). For the majority of the viruses, the coverage by contigs increased with subsample size, however, conversely, in several cases, it dropped at larger subsample sizes, i.e., TMV and PLMVd for sRNA and PepMV, CSNV, CaMV and CLVd for rRNA depleted totRNA approach (Figure ​ (Figure2D). 2D ). Contigs, assembled de novo from rRNA depleted totRNA datasets covered higher fractions of viral genomes for almost all viruses at all subsample sizes (coverage reached 95% at 10 million nts for majority of viruses), in comparison to sRNA derived contigs (95% coverage at 10 million nts was achieved only for PVY, TMV, and CLVd). Two exceptions to this observation were TYLCV and CLVd, for which sRNA derived de novo contigs cover higher genome fraction than rRNA depleted totRNA contigs, for all subsample sizes.

The comparison of the de novo assemblies for STV and ToMV revealed that when very low numbers of viral reads are recovered, the rRNA depleted totRNA approach is more effective, since in the case of the sRNA approach, no corresponding viral contigs were generated (Figure ​ (Figure2D). 2D ). A similar scenario was observed also for the putative novel Cytrohabovirus , where very low recovery of viral reads in the sRNA dataset resulted in no assembled contigs corresponding to this virus (Figure ​ (Figure2D 2D ).

In this study we compared the effectiveness of two NGS approaches that have been widely adopted for plant virus detection: sRNA deep sequencing and deep sequencing of rRNA depleted totRNA. When comparing the amount of virus/viroid reads recovered by one or the other approach, we observed different results for different viruses/viroids: in some cases, more viral/viroid nucleotides were recovered using sRNA and in other by rRNA depleted totRNA sequencing.

Detailed inspection of the results of the read mapping suggested higher recovery of virus reads for ssDNA viruses and viroids when using sRNA approach than when using rRNA depleted totRNA approach. For viroids, this could be the consequence of induced RNA silencing (Itaya et al., 2001 ; Papaefthimiou et al., 2001 ; Martínez de Alba et al., 2002 ) and, at the same time, the absence of the messenger RNA production, because, in the case of viroids, “long” RNAs are generated solely for the purpose of replication. Similarly, in the case of viruses with a circular ssDNA genome organization, a smaller fraction of viral nucleotides was recovered using rRNA depleted totRNA. In contrast with viruses with RNA genomes, for ssDNA viruses, RNA molecules are generated only during the transcription step, as messenger RNAs, which could be the reason for the lower recovery of viral nucleotides in this pool. Moreover, small RNAs could be amplified by the action of RNA-dependent RNA polymerase 6 (Borges and Martienssen, 2015 ) during the production of secondary sRNAs. The exception among the DNA viruses in this study was CaMV (DNA-RT), for which a higher fraction of virus nucleotides was recovered by sequencing rRNA depleted totRNA. The CaMV dsDNA genome is replicated through an RNA intermediary, in addition to producing messenger RNAs through transcription (Hull, 2014 ), which could explain a larger proportion of viral nucleotides in this pool.

All linear viruses in our infected plant samples had a ssRNA genome organization and synthesize different types of RNA throughout their replication cycle. For most of these viruses, sequencing rRNA depleted totRNA resulted in a larger proportion of reads mapping to the viral genomes (Figure ​ (Figure1) 1 ) compared with sRNA. However, a few exceptions were observed, PVY being the most notable with many more viral reads being present in the sRNA dataset. The high abundance of virus derived sRNA has already been reported for PVY (Kutnjak et al., 2015 ) and other potyviruses (Kreuze et al., 2009 ) even though they encode strong RNA silencing suppressors (Yelina et al., 2002 ; Ivanov et al., 2016 ).

In general, when read mapping was performed, 10 million nucleotides was sufficient to cover complete viral genomes using any of the two approaches (Figure ​ (Figure2C). 2C ). However, in some cases (STV and ToMV in sample III) very low numbers of viral reads were recovered (by both approaches), which negatively affected all the evaluated parameters. For those two cases, the percentage of virus reads (for both approaches) was lower than 0.1%, and the average read depth remained lower than 10×, and none of the viral genomes were completely covered by the reads even at the highest subsample size (50 million) (Figure ​ (Figure2C 2C ).

When comparing de novo assembly of sequencing reads, the rRNA depleted totRNA approach was generally more efficient than sRNA approach; this was demonstrated in higher proportion of viral genomes covered by de novo generated contigs from rRNA depleted totRNA datasets. The contigs assembled from rRNA depleted totRNA data covered at least a fraction of the consensus genome even in cases where the percentage of virus/viroid reads was lower than 0.1% and average depth lower than 10 (i.e., ToMV and STV) (Figure ​ (Figure2D). 2D ). In those cases, no viral contigs were assembled using sRNA datasets, probably due to a combination of low amount and small sizes of viral reads. Poorer coverage of viral genomes by sRNA derived de novo contigs is likely related to the more difficult assembly of very short sRNA reads into longer contigs, which has been observed previously (Kutnjak et al., 2015 ; Visser et al., 2016 ).

In some cases (PepMV, TMV, CSNV CaMV, CLVd, and PLMVd) smaller genome fractions are covered by contigs, when larger data sets are used for the assembly (corresponding to average depths > 100). This has been observed previously and is an artifact of the assembly algorithms (see CLC Analyses-related questions, 2017 ), which are not optimized for very high sequencing depths. After mapping reads or contigs to evaluate average depth and genome coverage (reads/contigs) we observed also the trend in generating higher or lower variability within 10 repeats. Unrepeatable random subsampling occurred when analysing smaller datasets and/or lower viral/viroid nucleotide proportion within the datasets, since all samples with this two features had greater variability.

The study has highlighted some points of difference between the compared approaches that may help to inform the choice of approach based on the purpose of the sequencing. This could be (i) screening against a list of known target organisms (e.g., at the import/export) and (ii) identification of the (possibly yet unknown) causal agent of the disease. Considering (i) screening against a list of known targets, this would be most cost effectively achieved using a method that maximizes the amount of viral sequences compared with host sequences. This study showed (Figures ​ (Figures1, 1 , ​ ,2A) 2A ) that the performance of the two compared approaches is very virus dependent. Broadly, sRNA performed better for circular ssDNA viruses and viroids, whilst rRNA depleted total RNA performed better for most of the tested linear RNA viruses with a notable exception (PVY). If considering (ii) sequencing for novel virus discovery, long contigs would provide the greatest chance of detecting very dissimilar sequences by comparing predicted amino-acid sequence from virus ORFs (e.g., with the use of BLASTx analysis or hidden Markov model based protein domain searches). The data shows that rRNA depleted total RNA generated longer contigs (which covered greater fractions of viral genomes) for most of the investigated viruses (Figure ​ (Figure2D). 2D ). As the most prominent example, an important difference between the compared approaches was observed on a case of a previously un-described Cytorhabdovirus , which was identified from the rRNA depleted total RNA following de novo assembly and BLASTx analysis, whilst the virus reads could only be found in the sRNA sequence data post-hoc (de novo assembly of sRNA reads did not generate any matching contigs).

The results of the comparison between the two NGS approaches highlight some trends that may guide diagnostic laboratories in the selection of a method appropriate for a specific application. However, whichever method is selected it is important to be aware of the limitations, some of which are detailed in this study, and follow up putative identification using an appropriate method. The recently published framework for handling novel plant viruses detected using NGS provides guidelines for achieving this (Massart et al., 2017 ).

In order to examine the potential costs of each method on commonly used Illumina sequencing platforms (HiSeq/sRNA and MiSeq/rRNA depleted totRNA) staff time used and reagent costs (in GBP) were calculated using list prices (Illumina) obtained on 1st March 2017. In general, both approaches generate more than sufficient amount of data than required to identify all of the viruses if mapping is used (50 million nts; Figure ​ Figure2). 2 ). HiSeq/sRNA sample will cost £138 and MiSeq/rRNA depleted totRNA sample will cost £159 if 24 samples (reasonable diagnostic throughput) are run per lane / flow cell, which is comparable price for output of 24 samples. Detail information about calculations is described in Supplementary data 1.2 and in Supplementary Table 6 .

The outcomes presented in this study showed that all included known viruses/viroids could be identified by both NGS approaches. Both approaches successfully identified also two divergent strains of PepMV, which was, despite short fragments of sRNA already shown previously (Kutnjak et al., 2014 ). However, a putative novel Cytorhabdovirus was only detected by analysing the data generated from ribosomal RNA depleted total RNA. Additionally, the results revealed the strength of NGS technology for the simultaneous detection and identification of several different known/unknown plant viruses from a different sample material, with a different amount of viral/viroid nucleotides and in a different host plants. Similar conclusions were derived from studies using other virus enrichment approaches on single or few viral species (Adams et al., 2009 ; Al Rwahnih et al., 2009 ; Kreuze et al., 2009 ; Kutnjak et al., 2014 ; Visser et al., 2016 ), e.g., both, sequencing of virion-associatad nucleic acids and sRNAs enabled a discovery of a new virus, previously overlooked by other detection techniques (Candresse et al., 2014 ). Our study further indicates the advantages of NGS in such cases and strengthens its use as a tool in plant virus/viroid diagnostics.

Author contributions

MR, DK, and NB conceived the idea, AP, MR, DK, and NB designed the experiments. AF provided samples. AP performed laboratory part of the experiment and analyzed the data with the assistance of IA and DK. AP wrote the draft of the manuscript. All authors significantly contributed with reviewing and editing the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Dr. Heiko Ziebell for providing the sample material, in this paper labeled as sample V, Pisum sativum infected with PNYDV, Dr. Ummey Hany for help with the library preparation and sequencing and Dr. Nataša Mehle for providing the sample material in this paper labeled as sample IX, Nicotiana benthamiana infected with CSNV.

Funding. The work was supported by COST Action FA1407 (DIVAS), thought STSM (short term scientific mission), Euphresco NGS-Detect project and Slovenian Research Agency, AP is a recipient of a Ph.D. research grant from the Slovenian Research Agency.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2017.01998/full#supplementary-material

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|     |     |      ID: Password:  | Author Resources Editor Resources Agents/Distributors     Current Topics in Virology Published in 2022 Original Article Pages  1 -  8 Amine Boudaoud, Bakir Mamache, Wafa Tombari, Jihene Lachhab, Abdeljelil Ghram, Nadir Alloui |   Original Article Pages  9 -  14 Dalale Behar, Lamia Boublenza, Bouchra Dahmani, Nafissa Chabni, Kassem Belatbi, Ikram Breik, Amel Benfoula, Ilyes Zatla, Hafida Hassaine |   and for detecting by nested RT-PCR Short Communication Pages  15 -  22 Susana A. Dandlen, Guilherme Russo, Soraia Horta, Amílcar Duarte, Paulo Paiva, Natália Marques | Short Communication Pages  23 -  28 Tatiana A. Timofeeva, Irina A. Rudneva, Aleksandr V. Lyashko, Irina M. Kupriyanova, Anastasia A. Treshchalina, Alexandra S. Gambaryan, Simone E. Adams, Galina K. Sadykova, Alexey G. Prilipov, Boris I. Timofeev, Natalia F. Lomakina | Original Article Pages  29 -  33 T. Valkov, G. Dimitrov, J. Hristova, G. Donkov, N. Yancheva-Petrova, R. Argirova |   Short Communication Pages  35 -  38 Thyyar M. Ravindranath |   Mini Review Pages  39 -  47 Martin Castelnovo, Fabien Montel, Cendrine Faivre-Moskalenko |   search E-Commerce Quick Links Browse in Alphabetical Order : Browse by Subject Classification : Miscellaneous Current Research in Virology Aims and scope. Current Research in Virology is ready to receive high quality research articles, CRV is dedicated to cover studies of viruses and virus-like agents, their structure, classification and evolution, their ways to infect and exploit cells for virus reproduction, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy. It is with great pleasure that we announce the SGAMR Annual Awards 2020. 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In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. View all journals Explore content About the journal Publish with us Sign up for alerts Review Article Published: 16 July 2019 Evolution and ecology of plant viruses Pierre Lefeuvre   ORCID: orcid.org/0000-0003-2645-8098 1 , Darren P. Martin 2 , Santiago F. Elena 3 , 4 , Dionne N. Shepherd 5 , Philippe Roumagnac   ORCID: orcid.org/0000-0001-5002-6039 6 , 7 & Arvind Varsani   ORCID: orcid.org/0000-0003-4111-2415 8 , 9   Nature Reviews Microbiology volume  17 ,  pages 632–644 ( 2019 ) Cite this article 11k Accesses 153 Citations 155 Altmetric Metrics details Microbial ecology Viral evolution Viral vectors Virus–host interactions The discovery of the first non-cellular infectious agent, later determined to be tobacco mosaic virus, paved the way for the field of virology. In the ensuing decades, research focused on discovering and eliminating viral threats to plant and animal health. However, recent conceptual and methodological revolutions have made it clear that viruses are not merely agents of destruction but essential components of global ecosystems. As plants make up over 80% of the biomass on Earth, plant viruses likely have a larger impact on ecosystem stability and function than viruses of other kingdoms. Besides preventing overgrowth of genetically homogeneous plant populations such as crop plants, some plant viruses might also promote the adaptation of their hosts to changing environments. However, estimates of the extent and frequencies of such mutualistic interactions remain controversial. In this Review, we focus on the origins of plant viruses and the evolution of interactions between these viruses and both their hosts and transmission vectors. We also identify currently unknown aspects of plant virus ecology and evolution that are of practical importance and that should be resolvable in the near future through viral metagenomics. This is a preview of subscription content, access via your institution Access options Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription 24,99 € / 30 days cancel any time Subscribe to this journal Receive 12 print issues and online access 195,33 € per year only 16,28 € per issue Buy this article Purchase on Springer Link Instant access to full article PDF Prices may be subject to local taxes which are calculated during checkout Similar content being viewed by others Vertical and horizontal transmission of plant viruses: two extremes of a continuum? Seasonality of interactions between a plant virus and its host during persistent infection in a natural environment Evolutionary dynamics of Tomato spotted wilt virus within and between alternate plant hosts and thrips Edwards, R. A. & Rohwer, F. Viral metagenomics. Nat. Rev. Microbiol. 3 , 504–510 (2005). CAS   PubMed   Google Scholar   Greninger, A. L. 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P.L. was supported by the European Union: European Regional Development Fund (ERDF), by the Conseil Régional de La Réunion and by the Centre de Coopération internationale en Recherche agronomique pour le Développement (CIRAD). S.F.E. was supported by a grant (BFU2015-65037-P) from Spain Ministry of Science, Innovation and Universities–ERDF. Author information Authors and affiliations. CIRAD, UMR PVBMT, St Pierre, La Réunion, France Pierre Lefeuvre Computational Biology Division, Department of Integrative Biomedical Sciences, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa Darren P. Martin Instituto de Biología Integrativa de Sistemas (I2SysBio), CSIC-UV, Paterna, València, Spain Santiago F. Elena The Santa Fe Institute, Santa Fe, NM, USA Research Office, University of Cape Town, Cape Town, South Africa Dionne N. Shepherd CIRAD, UMR BGPI, Montpellier, France Philippe Roumagnac BGPI, CIRAD, INRA, Montpellier SupAgro, University of Montpellier, Montpellier, France The Biodesign Center for Fundamental and Applied Microbiomics, Center for Evolution and Medicine, School of Life Sciences, Arizona State University, Tempe, AZ, USA Arvind Varsani Structural Biology Research Unit, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa You can also search for this author in PubMed   Google Scholar Contributions P.L., D.P.M., S.F.E., D.N.S., P.R. and A.V. wrote and edited the manuscript. P.L. and A.V. undertook the analyses for the data presented in figures 1–3. Corresponding author Correspondence to Arvind Varsani . Ethics declarations Competing interests. The authors declare no competing interests. Additional information Peer review information. Nature Reviews Microbiology thanks M. Roossinck, A. Whitfield and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Related links Plant viruses dataset: https://lefeup.github.io/plantviruses/ These invertebrate animals have exoskeletons, segmented bodies and paired jointed appendages. Arthropods belong to the phylum Euarthropoda that includes insects, arachnids, myriapods and crustaceans. These enzymes catalyse the synthesis of RNA from an RNA template. RNA-dependent RNA polymerases are essential to the replication of viruses that have no DNA stage. Some plant viruses encode these proteins to facilitate cell-to-cell movement of viral particles and/or uncoated viral nucleic acids. They frequently function by increasing the size exclusion limits of plasmodesmata. Brassica is a genus in the mustard family (Brassicaceae) of plants, which includes cabbage, lettuce and cauliflower. Angiosperms are also known as flowering plants and are the most diverse group of land plants. While both gymnosperms and angiosperms produce seeds, angiosperms are characterized by the presence of flowers, an endosperm within the seeds and the inclusion of seeds within fruits. These microscopic channels traverse plant cell walls enabling intercellular trafficking of macromolecules. This class of plant parasites comprises organisms in the orders Plasmodiophorida and Phagomyxida. They have long been recognized as a basal group to fungi, but recent molecular phylogenetic analysis suggests that they are more closely related to protozoa in the phylum Cercozoa. This order of insects includes insects such as aphids, cicadas, leafhoppers and planthoppers. Most hemipterans feed on plant sap with their sucking and piercing mouthparts. This is the body cavity in arthropods wherein haemolymph (plasma with haemocytes) circulates. These small sap-sucking insects are members of the superfamily Aphidoidea in the Hemiptera order. The phloem is the vascular system in plants within which soluble organic compounds that are produced during photosynthesis are transported. Rights and permissions Reprints and permissions About this article Cite this article. Lefeuvre, P., Martin, D.P., Elena, S.F. et al. Evolution and ecology of plant viruses. Nat Rev Microbiol 17 , 632–644 (2019). https://doi.org/10.1038/s41579-019-0232-3 Download citation Accepted : 13 June 2019 Published : 16 July 2019 Issue Date : October 2019 DOI : https://doi.org/10.1038/s41579-019-0232-3 Share this article Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. 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EDITORIAL article Editorial: microbial modulation to mitigate the impact of climate change on wine production. 1 Chemistry Research Centre–Vila Real (CQ-VR), Department of Agronomy, School of Agrarian and VeterinarySciences (ECAV), University of Trás-os-Montes and Alto Douro, Vila Real, Portugal 2 DAGRI-Department of Agriculture, Food, Environment and Forestry, University of Florence, Florence, Italy 3 EnotecUPM, ETSIAAB, Universidad Politécnica de Madrid, Madrid, Spain Editorial on the Research Topic Microbial modulation to mitigate the impact of climate change on wine production Microorganisms in vineyards and the surrounding soil can alter the composition of the final wine. The microbial community changes at the beginning of the winemaking process, and different types of wine yeasts dominate the grape juice and wine environment. Weather extremes related to climate change can disrupt the microbial balance in the wine, leading to undesirable characteristics in the final product. As winegrowers, winemakers, and scientists, your work is vital in preserving the quality of wine, especially in the face of climate change. The decrease in suitable viticulture areas and changes in grape composition present challenges. Many of you are studying yeasts and bacteria to mitigate these issues in warmer climates. Your work is significant and essential for improving wine quality through understanding and managing microorganisms in the vineyard and during winemaking. As winegrowers, winemakers, and scientists, you are not just at the forefront of mitigating the risks of climate change in the wine industry; you are also shaping its future. Recent advancements in 'omics' technologies have provided new opportunities for us to understand the grape/wine microbial ecosystem better. Specifically, unconventional non- Saccharomyces species, previously considered as spoilage microorganisms, are now recognized as beneficial as they enhance the wine aroma and taste when cultivated in controlled fermentations with Saccharomyces cerevisiae . Furthermore, ongoing biological approaches for modifying wine acidity using Saccharomyces and non- Saccharomyces yeasts and traditional lactic acid bacteria such as Oenococcus oeni and Lactiplantibacillus plantarum are being explored. This Research Topic explores how climate change can impact microbial diversity and subsequently alter wine characteristics. These risks can be mitigated by regulating the microbial community and utilizing yeast derivatives to enhance wine aroma and taste. Your work is not just important; it is empowering, as you are responsible for shaping the future of winemaking. This Research Topic comprises six types of work—one mini-review article, one review article, and four original research articles—written by international researchers to provide up-to-date research on the different dimensions of the vast research world of Microbial Modulation to Mitigate the Impact of Climate Change on Wine Production. The mini-review article ( Comuzzo et al. ) delves into emerging biotechnologies and non-thermal technologies for winemaking in the context of global warming. According to the authors, using non- Saccharomyces yeast species, such as Lachancea thermotolerans , can help manage some global warming wine issues by stabilizing pH and reducing alcohol content. Lower-pH wine improves freshness, palatability, and microbiological stability. Certain yeast species, like Hanseniaspora spp. and Metschnikowia pulcherrima , can enhance aroma complexity, improve the wine's sensory profile, and aid in acidification and bio-protection in winemaking. Bio-protection helps control oxidation, inhibit wild microorganisms, improve the implantation of starters, and limit SO 2 . Reductive yeast derivatives with high contents of reducing peptides and compounds like glutathione are also helpful in reducing SO 2 content. Also, emerging non-thermal technologies like Ultra High-Pressure Homogenization (UHPH) and Pulsed Light (PL) improve wine stability by controlling microbes and deactivating oxidative enzymes, which enhances the use of emerging non- Saccharomyces and reduces SO 2 additions ( Comuzzo et al. ). Puyo et al. studied the topic of bio-protection in enology by M. pulcherrima : From field results to a scientific inquiry. They wrote an interesting mini-review article, which mainly focuses on bio-protection using a non- Saccharomyces yeast, M. pulcherrima , recommended for bio-protecting grape musts. As they state, there are still many unanswered questions about the production of toxic compounds by M. pulcherrima , such as its potential production of killer toxins and its ability to make cell-cell contact interactions. The authors also point out that M. pulcherrima is known for consuming much oxygen. This high consumption could quickly deplete the oxygen in the grape must, preventing it from entering the pathways that lead to wine browning and producing unwanted aromas. Furthermore, M. pulcherrima secretes pulcherriminic acid, which chelates Fe 3+ once released into the medium. This ion is involved in redox mechanisms through the Fenton reaction. Reducing iron in the medium by pulcherriminic acid may also help, to a lesser extent, protect the grape-must from oxidation. This brief review examines the current state of field trials and laboratory studies demonstrating the effects of using yeasts for bio-protection and the interaction mechanisms responsible for these effects. M. pulcherrima was also the focus of the research article published by Torrellas et al. . The study delved into using non- Saccharomyces yeasts as starters in winemaking, namely the industrial problem of efficiently propagating this type of yeast. The work shows that the poor growth of Hanseniaspora vineae and M. pulcherrima in molasses is related to deficient sucrose consumption and low invertase activity. The authors modified the cultivation media to address this Research Topic, which involved hydrolysis and reducing the sucrose concentration. The results indicated that both species showed improved biomass production when cultivated in a hexose-based media, effectively addressing their low invertase activity. Reducing the sugar concentration also led to a respiratory metabolism, resulting in a higher biomass yield. However, the modifications did not enhance biomass production due to reduced sugar availability. To assess the effectiveness of these changes, fermentations using mixed grape juice with biomass produced under conditions similar to M. pulcherrima and S. cerevisiae were conducted. The analysis of the resulting wines indicated that the treatments tested did not negatively impact wine quality, demonstrating their potential for practical application on an industrial scale to improve biomass production. In an original research article, Vion et al. explored the intracellular metabolic variations between seventeen S. cerevisiae strains belonging to two different genetic populations, flor yeasts and wine yeasts, in alcoholic fermentation. These two populations are closely related, share the same ecological niche, and have distinct genetic characteristics. The authors developed a 1H-NMR protocol to measure the intracellular concentration of yeast biomass during the alcoholic fermentation of natural grape juice. The protocol was used to analyze the different metabolic contents of several S. cerevisiae strains (flor and wine yeasts). Additionally, accurate quantification of 21 metabolites in a two-time series provided new results that showed that intracellular metabolic variability is influenced by the sampling time and the yeast strain, with complex interactions that prevent simple physiological conclusions. Dournes et al. in an original research work, focused on the question of copper's impact on varietal thiols in wine, taking into account the use of this compound in organic vineyard management as the sole fungal control pesticide against downy mildew. Colombard and Gros Manseng grape juices were fermented under different copper levels to mimic the consequences in grape-must of organic practices. The authors found that the highest copper level for both grape varieties significantly increased yeast consumption of precursors. Also, for both grape varieties, free thiol content in wine significantly decreased. The amount of thiol produced during fermentation remained constant for Colombard grapes, regardless of copper levels, indicating that copper only had an oxidative effect on this variety. However, during Gros Manseng fermentation, the thiol content increased as the copper content increased, showing a potential 90% increase. This suggests that copper might alter the pathways for producing varietal thiols, highlighting the significant role of oxidation. These findings contribute to understanding how copper affects thiol-focused fermentation and emphasize the importance of considering total thiol production (both reduced and oxidized) to comprehend the impact of studied factors better and distinguish between chemical and biological effects. Considering the importance of yeast co-inoculations in winemaking, which aims to modulate the aromatic profiles of wines, Bordet et al. investigated the impact of three co-cultures and corresponding pure cultures of S. cerevisiae on the chemical composition and sensory profile of Chardonnay wine. The use of co-cultures affected esters, fatty acids, and phenol families. Comparisons between the sensory profiles and metabolomes of co-cultures, pure cultures, and associated wine blends have revealed distinct differences. It was observed that the co-culture did not simply combine the characteristics of the two pure-culture wines, indicating an interaction effect. High-resolution mass spectrometry identified thousands of biomarkers specific to the co-cultures. Additionally, the study highlighted the involvement of metabolic pathways, particularly those related to nitrogen metabolism, in the observed changes in wine composition. The authors concluded that mixed S. cerevisiae yeasts can modulate the aromatic and chemical profile of wines without affecting their fermentative properties. They found that traditional methods, like monitoring yeast populations over time, are insufficient for understanding the interactions between yeast strains. A comprehensive approach involving different techniques is necessary to comprehend these interactions fully. In summary, the Research Topic “ Microbial modulation to mitigate the impact of climate change on wine production ” demonstrates that microbial modulation can mitigate the impact of climate change on wine production using different techniques, yeast strains, and biochemical approaches. Moreover, when conjugated with cutting-edge analytical methods, biotechnologies, and non-thermal technologies, it helps improve wine quality and safety. Author contributions AV: Writing – original draft, Writing – review & editing, Conceptualization, Project administration. PD: Writing – review & editing, Conceptualization, Project administration, Validation. AM: Writing – review & editing, Conceptualization, Project administration, Validation. The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Acknowledgments AV greatly indebted to the authors who generously shared their scientific knowledge and experience with others through their contribution to this Research Topic. AV incredibly thankful to AM and PD for embracing this small adventure with me. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Keywords: non-saccharomyces yeasts, mixed-culture fermentations on wine quality, biotechnological strategies to improve wine production and safety, bioprotection and sulfur dioxide reduction, non-thermal technologies Citation: Vilela A, Domizio P and Morata A (2024) Editorial: Microbial modulation to mitigate the impact of climate change on wine production. Front. Microbiol. 15:1465637. doi: 10.3389/fmicb.2024.1465637 Received: 16 July 2024; Accepted: 23 July 2024; Published: 07 August 2024. Edited and reviewed by: Giovanna Suzzi , University of Teramo, Italy Copyright © 2024 Vilela, Domizio and Morata. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Alice Vilela, avimoura@utad.pt Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. 269 IMAGES Encyclopedia of Virology: New Research (6 Volume Set) The major topics of this review are summarized. 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Research in Virology 5th Rhine-Main Symposium on HIV and Macrophages. View all issues. Read the latest articles of Research in Virology at ScienceDirect.com, Elsevier's leading platform of peer-reviewed scholarly literature. Current Opinion in Virology Current Opinion in Virology (COVIRO) is a systematic review journal that aims to provide specialists with a unique and educational platform to keep up to date with the expanding volume of information published …. View full aims & scope. $3480. Article publishing charge. Current Research in Virological Science Read the latest articles of Current Research in Virological Science at ScienceDirect.com, Elsevier's leading platform of peer-reviewed scholarly literature Home page Virology Journal is an open access, peer reviewed journal that considers articles on all aspects of virology, including research on the viruses of animals, plants and microbes. The journal welcomes basic research as well as pre-clinical and clinical studies of novel diagnostic tools, vaccines and anti-viral therapies. Read more. Virology Deadly diseases and inflatable suits: how I found my niche in virology research Virologist Hulda Jónsdóttir studies some of the world's most pathogenic viruses at the Spiez Laboratory in ... Virology News, Articles and Research News-Medical is your trusted source of Virology news, articles and research for doctors, patients, and families. Next Generation Sequencing for Detection and Discovery of Plant Viruses Diagnosis of plant viruses using next-generation sequencing and metagenomic analysis, in Current Research Topics in Plant Virology, eds Wang A., Zhou X. (Cham: Springer; ), 323-335. 10.1007/978-3-319-32919-2_14 [ CrossRef] [ Google Scholar] Current Research Topics in Plant Virology Topics covered in this book include RNA silencing and its suppression in plant virus infection, virus replication mechanisms, the association of cellular membranes with virus replication and movement, plant genetic resistance to viruses, viral cell-to-cell spread, long distance movement in plants, virus induced ER stress, virus diversity and ... Current Topics in Virology 1. Molecular characterization of wild infectious bursal disease viruses (IBDV) that are implicated in vaccination failures in North East of Algeria. Original Article. Pages 1 - 8. Amine Boudaoud, Bakir Mamache, Wafa Tombari, Jihene Lachhab, Abdeljelil Ghram, Nadir Alloui. Abstract | Buy this article. Current Research in Virology Aims and Scope. Current Research in Virology is ready to receive high quality research articles, CRV is dedicated to cover studies of viruses and virus-like agents, their structure, classification and evolution, their ways to infect and exploit cells for virus reproduction, the diseases they cause, the techniques to isolate and culture them ... Systems virology Systems virology is the scientific discipline that integrates high-throughput molecular techniques and computational tools to study all aspects of viruses and viral diseases, in an effort to ... The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins The US has one of the strongest and safest infrastructures for research globally. The policies aimed at virology research in the US will not protect against work with viruses of known pandemic potential occurring at inadequate biosafety containment (below biosafety level 3) in other countries, which poses the risk of lab exposures. Researchers create new treatment and vaccine for flu and various Researchers have discovered new ways of preventing and treating respiratory viruses. In two new papers, the team reports the development and validation of NanoSTING, a nasal spray, as a broad ... Virology: Current Research- Open Access Journals Virology: Current Research is an open access peer reviewed journal initiated by Hilaris SRL aims to provide cutting-edge research findings in the field of virology. The journal offers broad coverage to the researchers those who are interested in virology and related fields. Virology: Current Research an avenue and provide a forum for the ... SARS-CoV-2 SARS-CoV-2 articles from across Nature Portfolio SARS-CoV-2 is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19). Evolution and ecology of plant viruses The pervasive perception that plant viruses are primarily pathogens has meant that most plant virology research has focused on the causes and consequences of virus pathology. Frontiers This Research Topic comprises six types of work—one mini-review article, one review article, and four original research articles—written by international researchers to provide up-to-date research on the different dimensions of the vast research world of Microbial Modulation to Mitigate the Impact of Climate Change on Wine Production. assignment essay homework paper phd report resume review speech thesis