Plant viruses have always been a challenge to plant growth and crop production in several parts of the world. Virus can be transmitted by vegetative propagation, fungi, nematodes, aphids, leaf hoppers, plant hoppers, beetles, white flies, and so forth. Viruses symptoms vary with the infecting virus and the infected part which includes leaf spots, leaf blights, root rots, fruit rots, fruit spots, wilt, dieback and decline. It is causing economic losses by reducing crop quality, quantity and nutritional value. Thus, their reliable detection is of a crucial importance for plant protection. While the adoption of molecular techniques such as RT-PCR has increased the speed and accuracy of virus diagnostics, such techniques only allow the detection of known viruses, i.e., each test is specific to one or a small number of related viruses. Therefore, unknown virus can be missed and testing can be slow and expensive if molecular tests are unavailable. NGS technology is one of the most popular tools for virus diagnostics. It is highly efficient, rapid diagnostics tools, and low-cost high-throughput and deep RNA sequencing. Due to the capacity to target multiple unique signature loci of virus in an infected plant metagenome and also useful for discovery of new virus and new hosts. It is including virus genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. By using deep RNA-seq requires fast and robust bioinformatics methods to enable host sequence removal and virus classification. Future developments in this area, including the use of bioinformatics tools for identification and characterization of multiple plant virus and analysis of diversity of plant viruses.

1. Next generation sequencing for Identification
and Characterization of plant viruses
SUBMITTED BY-
Malyaj R Prajapati
Ph.D. Plant Molecular Biology and Biotechnology
College of Biotechnology
SVBPUA&T, MEERUT

2. Plant Virus
 Plants have specific viruses.
 Viruses cause many plant diseases.
 They are responsible for losses in crop yield and quality.
 Viroids are infectious RNAs that cause important plant
diseases.
 These pathogens are similar to some plant viruses in that they
contain an RNA genome.
 They differ from RNA plant viruses that they are composed of
naked RNAs and lack a protein coat.
 Viroids do not produce any proteins when they infect a plant
cell despite the fact that they are made of RNA.

3. Symptoms of Plant Virus

4. What is Next Generation Sequencing?
High-throughput sequencing
This technology allow for sequencing of DNA and RNA much
more quickly and cheaply than the previously used Sanger
sequencing, and as such revolutionized the study of genomics and
molecular biology.
Advantages of NGS
NGS can be used to analyse DNA and RNA samples and is a popular tool in
functional genomics.
 a priori knowledge of the genome or genomic features is not required
 it offers single-nucleotide resolution, making it possible to detect
related genes (or features), alternatively spliced transcripts, allelic gene
variants and single nucleotide polymorphisms
 higher dynamic range of signal
 requires less DNA/RNA as input (nanograms of materials are
sufficient)
 higher reproducibility

5. •Illumina (Solexa) sequencing:
Illumina sequencing works by simultaneously identifying
DNA bases, as each base emits a unique fluorescent signal,
and adding them to a nucleic acid chain
•Roche 454 sequencing:
This method is based on pyrosequencing, a technique which
detects pyrophosphate release, again using fluorescence, after
nucleotides are incorporated by polymerase to a new strand
of DNA.
•Ion Torrent: Proton / PGM sequencing:
Ion Torrent sequencing measures the direct release of H+
(protons) from the incorporation of individual bases by DNA
polymerase and therefore differs from the previous two
methods as it does not measure light.

6. Next Generation Sequencing Platform

7. Infected
Sample
Nucleic acid
template
Library
preparation
HTS
Denovo
Assembly
Quality
trimming
Raw reads Virus contigs
Contig
annotation
Motif detection
Genome
organization
Sanger
sequencing
PCR validation
Validation by
specific primer
Homology search
Workflow for Identification and Characterization of Plant Virus
Phylogenetic
analysis
Recombination
detection
RDP

8. Different approaches of RNA Isolation
 Total nucleic acid
 This approach has the advantage of not biasing
the results in favour or against any particular
virus and allows for simple sample processing
which reduces costs.
 This method was first used by Rwahnih et al.
(2009) and Adams et al. (2009).
 Recently used of this identify a large range of
viruses in carrots (Rubio et al., 2020).
Enrichment
NGS
platform
Assembly
tools
Viruses Viroids
Host species
Known
New
Known New
RNA DNA
Total RNA 454 CLC 3 1 3 Grapevine
454 CLC 1 1 Tomato/Gom
phrena
globosa
454 CLC 1 Cassava
454 CLC 1 Canna
Illumina Geneious 1 Hardenbergia
comptoniana
Illumina Velvet/Gene
ious
1 H.
comptoniana
454 CLC 1 Lettuce
454 CLC 1 Iris
Illumina Geneious/C
LC
1 Yellow
tailflower
Illumina Geneious 1 Capsicum
annuum

9.  Ribosome depletion
 A modification of the total RNA method is to use
one of the ribosome subtraction technologies to
remove the plant ribosomal RNA from the total
RNA.
Enrichment
NGS
platform
Assembly
tools
Viruses Viroids
Host species
Known
New
Known New
RNA DNA
rRNA
depletion
Illumina 3 4 Grapevine
Illumina
Hiseq 2000
CLC 9 1 Garlic

10.  Double Stranded (ds)RNA
 In this apporoch possible to purify dsRNA using
cellulose (Dodds et al. 1984) or using lithium
chloride (Akin et al. 1998).
 Using this method grapevine viruses were sequenced
Rwahnih et al. (2009). It has since been used
successfully many times (Roossinck et al. 2015;
Barba et al. 2014).
 DMSO treatment improved sequencing read
recovery by over two orders of magnitude, even
when RNA and cDNA concentrations were below
the limit of detection. Barba et al. 2014 were tested
the effects of DMSO on a mock eukaryotic viral
community and found that dsRNA virus reads
increased with DMSO treatment.
Enrichment
NGS
platform
Assembly
tools
Viruses Viroids
Host species
Known
New
Known New
RNA DNA
dsRNA 454 CLC 3 1 3 Grapevine
Illumina Velvet 4 Grapevine
454 Newbler 1 Grapevine
Illumina Velvet/
CodonCode
1 Red raspberry
454 CLC 1 Cherry
Illumina Velvet 1 Grapevine
454 Newbler 2 Maize
Illumina Geneious/
CLC
1 Orchid
Illumina Velvet 1 Grapevine
Illumina Velvet 2 Japanese
persimmon
Illumina Velvet PVd2 Diospyros
virginiana
Illumina CLC 1 Citrus
Illumina Geneious/
CLC
10 Garlic

11.  Small RNA
 The plant detects viral dsRNA and uses
the enzyme dicer to cleave the dsRNA
into small 21–24 nucleotide fragments.
 These small RNA molecules are called
small interfering RNAs.
 Kreuze et al. (2009) identified a series of
plant viruses by purifying and sequencing
plant siRNA.
 The method has since been used
successfully to sequence a whole range of
viruses (Barba et al. 2014; Roossinck et
al. 2015).
Enrichment
NGS
platform
Assembly
tools
Viruses Viroids
Host species
Known
New
Known New
RNA DNA
Small
RNAs
Illumina Velvet/VCAK
E
2 3 Sweet potato
454 Velvet 2 Cocksfoot grass
Illumina Blast 12 Grapevine
Illumina SSAKE 27 1 Grapevine
Illumina Velvet 1 Sweet potato
Illumina Velvet 2 Tomato
Illumina Velvet 1 Squash
Illumina Velvet 6 Sweet potato
Illumina Velvet 1 1 1 Tomato
Illumina Velvet 3 1 2 Grapevine
Illumina Velvet 1 2 Citrus
Illumina Velvet 1 Citrus
Illumina Velvet 1 Yam bean
Illumina Velvet 10 1 Arctium tomentosum
Illumina Velvet 1 Soybean
Illumina Velvet 1 Sweet potato
Illumina Velvet 1 1 Potato
Illumina Velvet 1 Tomato
Illumina Velvet/Oases 1 Citrus
Illumina Velvet/Oases 1 Citrus
Illumina CLC 1 1 Sugarcane
Illumina CodonCode 1 Papaya
Illumina Geneious/Vel
vet/
CLC/Geneious
3 1 Tomato
Illumina Velvet 19 Sweet potato
Illumina Velvet 1 Potato
Illumina Velvet 1 2 Grapevine
Illumina Velvet 1 3 Cassava
Illumina Velvet 1 Pagoda
Illumina Velvet/CAP3 3 1 Rose

12. NGS based identification of viruses strongly associated with diseases of agricultural crops

13. Garlic (Alium sativum)
 The viral content of garlic has been analyzed using RNA-seq
to make the case for using multiplex methods for virus
detection in the context of plant quarantine systems (Wylie
et al., 2014).
 Total RNA was extracted from leaves from garlic plants,
amplified and sequenced using Illumina HiSeq 2000
technology (Singh et al., 2020).
 More than 40 virus isolates were identified including
potyviruses (e.g., Leek yellow stripe virus), allexiviruses
(e.g., Garlic virus D (GarVD), Garlic virus X (GarV-X) and
carlaviruses (e.g., Shallot latent virus) (Singh et al., 2020).

14. Pepper (Capsicum annuum)
 Multiple viral infections have also been identified in
pepper plants (Capsicum annuum) using RNA-seq
analysis (Jo et al., 2017).
 More than 10 viruses were identified, including Bell
pepper endornavirus (BPEV), PepLCBV (Pepper leaf
curl Bangladesh virus), and TVCV (Tobacco vein
clearing virus).
 And a novel virus, Pepper Virus A (PepVA) were
identified (Jones et al., 2017).

15. Grapevine (Vitis vinifera)
 Grapevines are the most well-known plant host, with more
than 64 viruses, including viroids (Maliogka et al., 2015).
 The extraction of virus-enriched RNAs followed by NGS can
identify several viruses and viroids infecting grapevines.
 The viruses identified included Grapevine leafroll-associated
virus 3 (GLRaV-3), Grapevine rupestris stem pitting-
associated virus (GRSPaV) and Grape vine virus A (GVA).
Grapevine virus E.
 The most prevalent viruses identified were Grapevine yellow
speckle viroid 1 (GYSVd1), Grapevine pinot gris virus
(GPGV), Hop stunt viroid (HSVd), and Grapevine leafroll-
associated virus 2 (GLRaV2) (Jones et al., 2017).

16. Tomato (Solanum lycopersicum)
 Viral infection is one of the major factors limiting tomato production.
 22 viruses were identified, including both know and newly detected viruses by using
small RNA-seq (Xu et al., 2017).
 Tomato infecting viruses:
Tomato mosaic virus (ToMV, genus Tobamovirus), Tomato yellow leaf curl
virus (TYLCV, genus Begomovirus), Potato virus Y (PVY, genus Potyvirus), Southern
tomato virus (STV, genus Amalgavirus), Cucumber mosaic virus (CMV,
genus Cucumovirus), Chilli veinal mottle virus (ChiVMV, genus Potyvirus), Tomato
mottle mosaic virus (ToMMV, genus Tobamovirus), Tomato chlorosis virus (ToCV,
genus Crinivirus), Tomato zonate spot virus (TZSV, genus Tospovirus), and Tomato
spotted wilt virus (TSWV, genus Tospovirus)

17. Novel plant viruses discovered by next-generation sequencing
Gayfeather mild mottle virus (GMMV) Bromoviridae (Cucumovirus)
Grapevine Syrah virus-1 (GSyV-1) Tymoviridae (Marafivirus)
Cassava brown streak virus (CBSV) Potyviridae (Ipomovirus)
Hardenbergia virus (HarVA) Betaflexiviridae
Lettuce necrotic leaf curl virus (LNLCV) Secoviridae (Torradovirus)
Yellow tailflower mild mottle virus (YTMMV) Virgaviridae (Tobamovirus)
Caladenia virus A (CalVA) Potyviridae (Poacevirus)
Donkey orchid virus A (DOVA)
Diuris virus A (DiVA)
Diuris virus B (DiVB)
Diuris pendunculata cryptic virus (DPCV)
Potyviridae (Potyvirus)
Betaflexiviridae (Capillovirus) Betaflexiviridae (Capillovirus)
Partitiviridae
Ipomoea batatas Rhabdovirus N-like sequences
(IbRNLS)
Cymbidium mosaic virus (CymMV)
Rhabdoviridae
Alphaflexiviridae (Potexvirus)
Raspberry latent virus (RpLV) Reoviridae (Reovirus)
Grapevine virus F (GVF) Betaflexiviridae (Vitivirus)
Donkey orchid symptomless virus (DOSV) Unassigned
Persimmon virus A (PeVA)
Persimmon latent virus (PeLV)
Rhabdoviridae (Cytorhabdovirus)
Unassigned
Eggplant mild leaf mottle virus (EMLMV) Potyviridae
Pepper yellow leaf curl virus (PYLCV) Luteoviridae (Polerovirus)
Tomato necrotic stunt virus (ToNSV) Potyviridae (Potyvirus)
Grapevine pinot gris virus (GPGV) Betaflexiviridae (Trichovirus)
Citrus yellow vein clearing virus Y1 (CYVCV-Y1) Alphaflexiviridae (Mandarivirus)
Yam bean mosaic virus (YBMV) Potyviridae (Potyvirus)
Woolly burdock yellow vein virus (WBYVV) Unassigned
Sweet potato C6 virus (SPC6V) Betaflexiviridae (Carlavirus)
Andean potato mild mosaic virus (APMMV) Tymoviridae (Tymovirus)
Tomato mottle mosaic virus (ToMMV) Virgaviridae (Tobamovirus)
Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) Cilevirus
Citrus vein enation virus (CVEV) Luteoviridae (Enamovirus)
Tomato matilda virus (TMaV) Iflaviridae (Tomavirus)
Cassava polero-like virus (CsPLV)
Cassava new alphaflexivirus (CsNAV)
Cassava torrado-like virus (CsTLV)
Luteoviridae (Polerovirus)
Alphaflexiviridae (Potexvirus)
Secoviridae (Torradovirus)

18. Sweet potato badnavirus C1 (SPBV-C1)
Sweet potato badnavirus C2 (SPBV-C2)
Caulimoviridae (Badnavirus)
Caulimoviridae (Badnavirus)
Grapevine red blotch-associated virus (GRBaV) Geminiviridae
Sugarcane white streak virus (SWSV) Geminiviridae (Mastrevirus)
badnaviruses
mastrevirus
Caulimoviridae (Badnavirus)
Geminiviridae (Mastrevirus)
Grapevine vein clearing virus (GVCV) Caulimoviridae (Madnavirus)
Citrus chlorotic dwarf-associated virus (CCDaV) Geminiviridae
Grapevine geminivirus (GVGV) Geminiviridae
Pagoda yellow mosaic associated virus (PYMAV) Caulimoviridae (Badnavirus)
DNA virus
(Wu et al., 2016)

19. MT731489_GarV-A_Meerut
MN059393_GarV-D_China
MN059327_GarV-E_China
AJ292230_GsrV-E_China:Zhejiang
MK503771_GarV-X_India
MN059429_GarV-X_China
U89243_GarV-X_Korea
MN059429_GarV-X_China
NC_025789_GarV-B_Argentina
KM379144_GarV-B_Argentina
MN059179_GarV-B_China
MK518067_GarV-D_India
a)
b
)
Characterization and Phylogenetic study of Plant Virus

20. Tools for Plant virome study

21. In the near future, as NGS becomes a well-established technique, both
methodologies and analysis pipelines should be harmonized among research
laboratories.
Technological advances and increased competition will continue to push the field
towards lower costs, higher throughput and more user friendly options for analysis.
Therefore, NGS technologies are becoming an affordable and promising means to
explore many plant virology and to develop appropriate prevention aids so as to
improve their trade potential and food security.
Future Perspectives

22.  The power of next-generation sequencing (NGS) technologies to allow rapid determination of
the total nucleic acid content in a biological sample has transformed the diagnosis and
identification of Plant viruses.
 All viruses and viroids identical or similar to those described previously can be identified in a
plant sample by a single NGS run.
 More than 50 new viruses has been identified by NGS since 2009.
 Common strategies to enrich viruses and/or viroids for deep sequencing include the
purification of dsRNAs, virus-like particles, or small RNAs. However, identification of RNA
and DNA viruses in a single NGS run is possible only by sequencing total small RNAs.
Importantly, the method could be used for the study of:
 Plant–virus–vector interactions through transcriptomic analyses to unveiling mechanisms
of pathogenesis and disease development.
 Studies of resistance mechanisms, such as RNA silencing, acting against viruses in
different plant species, which in the end, could lead to the development of novel control
tools.
 NGS has applications in genetic diversity, small RNA/gene expression and epidemiological
studies
Conclusion

23. References
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• Jones, S., Baizan-Edge, A., MacFarlane, S., & Torrance, L. (2017). Viral diagnostics in plants using next generation sequencing:
computational analysis in practice. Frontiers in plant science, 8, 1770.
• Rubio, L., Galipienso, L., & Ferriol, I. (2020). Detection of plant viruses and disease management: relevance of genetic diversity and
evolution. Frontiers in plant science, 11, 1092.
• Syller, J. (2012). Facilitative and antagonistic interactions between plant viruses in mixed infections. Molecular plant
pathology, 13(2), 204-216.
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dependent and homology-independent algorithms. Annual review of phytopathology, 53, 425-444.
• Adams, I. P., Skelton, A., Macarthur, R., Hodges, T., Hinds, H., Flint, L., & Fox, A. (2014). Carrot yellow leaf virus is associated with
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25. Thank You