Virus NGS

1. virus By Mesele Tilahun Belete
University of Science and Technology, Korean Research Institute
of Bioscience and Biotechnology (KRIBB) Campus
Supervisors: Moon Jae Sun (Ph.D.)
Course Instructor: Yoo Hee-min (Ph.D.)
Course name: Biological Analysis
Biotechnology Seminar
Oct 12, 2022
KRISS-UST

3. OUTLINES
 General Introduction
 Materials and Methods
 Result and discussion
 Conclusion
 Future study
2
OUTLINES

4.  Important tobacco diseases & caused by multiple causal agents.
 first reported in Zimbabwe,
 It was reported that the TBTD in China was caused by
 The discovery of a so‐far unknown virus is a rare and exciting event
The work described with such a discovery:
The causal agent of disease in tobacco plants appeared to be a plant virus that was not
described before
Tobacco bushy top disease (TBTD)
 Tobacco bushy top virus (TBTV),
 Tobacco vein distorting virus (TVDV),
 Tobacco bushy top virus satellite RNA (TBTVsatRNA)
 Tobacco vein distorting virus associated RNA
(TVDVaRNA).
 The occurrence of TBTD first recorded in 1993 in Yunnan, China.
 Up to 2001 51,300 ha crop was infected and harvest losses were
estimated to exceed US$ 33M
General Introduction

5. Systemic symptoms
 Systemic chlorosis
 Vein clearing, Vein banding
 Mosaic, stripes, streaks, Mottle
 Leaf Yellowing and distortion
 Deformations (leaf-, flower)
 Necrosis (leaf, stem, terminal bud)
 Flower breaking, Wilt
 Stunting , dwarfing
Local symptoms
 Local chloroses
 Local necroses, HR
 Starch lesions
Figure 1. Symptoms of TBTD-affected flue-cured tobacco cultivar K326 in field.
A-Symptoms of an early infection (chlorosis, internode shortening & serious stunting)
B- Early infected (indicated by the red arrow) and healthy looking tobacco plants
C- Late infection symptoms (small leaves, foliar yellowing or chlorosis, stunting).
D- Late infected plant (left) and healthy looking tobacco plant (right)
 The best disease prevention and management strategies
rely on knowledge of the viruses and their vectors including
 Biological properties and epidemiology,
 Recourse to biological & chemical control methods
Features
DNA :
RNA :
- ssDNA (rare) eg. Geminivirus
- dsDNA (15-20%) eg. Caulimovirus
- ssRNA (+ /-) (70-80%) eg. Tobamovirus (+),Tospovirus(-)
- dsRNA (rare) eg. Reovirus
General Introduction

6. https://www.frontiersin.org/articles/10.3389/fmicb.2019.01957/full
Plant viral
detection
methods
Pure culture
Nucleic acid based
PCR variants
-multiplex PCR
-Immuno-PCR
-SISPA
-Real Time PCR
-Nanofludic digital PCR
Isothermal amplification
-Paper-LAMP
-NASBA
-SPIA
-RPA
-PSR
-TaqMan probe PCR
Next generation methods
-Mass spectrometry
-NGS
-Biosensor
-Microarray
-Aptamer
Prospective techniques
-Droplet digital/ PCR
-NASBA-CRISPER
-Peptido nucleic acid
Electron microscopy
-Cryo-EM
-SP-EM
Serological
-LAT & ICT
-ELISA
-Bioluminescent EIA
SDS-PAGE
Western blot
 NGS don’t require prior knowledge potential risk outbreak in many crop species
 detection of novel viruses, frequently infected virus
Determine global virus diversity existing in plant communities & ecosystems
They depend on prior knowledge
(antibody and sequence)
morphological
PCR
Enzyme-linked immunosorbent assay (ELISA)
General Introduction

7.  Two tobacco plants: YBSh and YKMPL showing TBTD symptoms were
collected in 2015 and 2016 respectively, and were stored in insect-proof
greenhouse.
Field samples collection
Table 1. Sample ID of Crofon weed, Purple Perilla & Dahlia.
 1550 virus-like leaf samples from plants 29 species
(817 tobacco leaf samples + 733 leaf samples from 29 plant species)
 65 pepper plants
 83 tomato plants
 11 crofon weed leaf
 3 dahlia
 To survey the occurrence of TBTV, TVDV, TBTVsatRNA and TVDVaRNA in fields,
YBSh : Yunnan Baoshan
YKMPL: Yunnan Kunming
Materials and Methods

8. YBSh and YKMPL
Sample collection
Total RNA extraction
Library
preparation
Illumina HiSeq X-ten
Platform NGS
Materials
Data
analysis
Obtained raw
reads High-quality reads
De novo assembly
by Trinity program
BLAST searches
Confirm
Identify known
virus seq-fragments
Design primers to
join fragments
Confirm by RT-PCR using
virus-specifc primers
Sanger Sequencing
rRNA
Low quality & adapters
Phylogenetic
analysis
NGS experiment setup
-Sorted out as candidate genomic
fragments of the novel virus
RACE
Assembled
Predict ORF
comparisons
Epicentre RiboZeroTM kit
Materials and Methods

9. YBSh YKMPL
Total clean RNA reads 36,744,321 33,356,805
Assembled contigs>200 56,964 52,916
TBTVsatRNA 122,028 reads 116,779 reads
TVDVaRNA 22,370 reads 1124 reads
TBTV 1775 reads 14,388 reads
TVDV 15,430 reads 2923 reads
TPV1 6397 reads 277 reads
TPV2 8594 reads 339 reads
 The results showed that both the YBSh and YKMPL tobacco
samples were co-infected with 6 different viral agents
Tobacco bushy top virus (TBTV)
 genus Umbravirus, family Tombusviridae
 +ssRNA viruses
 does not have a 5′ cap and a 3′ poly(A) tail
 does not encode a capsid protein (CP)
 doesn’t form typical virion structure
 need helper viruses, mostly a polerovirus
or an enamovirus for transmission
Tobacco vein distorting virus (TVDV
 is a member in the genus Polerovirus,
family Solemoviridae
Total RNAs (817 tobacco leaf samples)
PrimeScript™ One-Step RT-PCR Kit
cloned into the pMD19-T vector
sequenced by BGI (China)
assembled by DNASTAR 7.0 package
 Sequence confirmation of 2 new
poleroviruses in the field tobacco
samples
 Differences in aa sequence identity of any gene product of >10%
Result and discussion

10. TBTV- tobacco bushy top virus
TVDV-tobacco vein distorting virus
TBTVsatRNA-
TVDVaRNA -
specific virus amplicons are indicated with arrows.
H: means healthy tobacco plant,
YBSh and YKMPL indicate the two TBTD samples.
Figure S2. Amplification strategies for TBTV, TVDV and TVDVaRNA genomes
detected by conventional RT-PCR
TPV1 TPV2
Figure S1
detected by multiplex one-step RT-PCR
 Based on the results of sequence alignment, two new tobacco polerovirus 1
(TPV1) and tobacco polerovirus 2 (TPV2) obtained.
 To validate the reliability of HTS results, the total RNA samples isolated from the
YBSh, YKMPL samples as well as healthy tobacco sample were analyzed by RT-
PCR.
 TPV1 and TPV2 detection primers were designed according to the virus contigs
identified through HTS, while the primers and multiplex one-step RT-PCR used to
detect TBTV, TBTVsatRNA, TVDV, TVDVaRNA .
 The results showed that the PCR products representing the six viruses were indeed
present in the YBSh and YKMPL samples, but not in the sample from healthy
plant.
Result and discussion

11. Figure 2. Genome structures of TPV1, TPV2 and PLRV
Result and discussion

12. tobacco virus 2
sugarcane yellow leaf virus
Table 2. Nucleotide sequence comparisons using the nearly full-
length genomic sequences of TPV1 with that of other poleroviruses.
Result and discussion
Table 3. Nucleotide and aa sequence comparisons of diferent ORFs of TPV1 with
those of other poleroviruses.

13. Table 4. Nucleotide sequence comparisons using the near full
genomic sequence of TPV2 with that of other poleroviruses. Table 5. Nucleotide and aa sequence comparisons of different ORFs of TPV2
with those of other poleroviruses.

14. Table 6. RT-PCR detection results for TPV1 and TPV2 from 244 feld tobacco samples.
Table 7. RT-PCR detection of TPV1, TPV2 and the four TBTD causal viruses in 33 field collected tobacco samples.
Result and discussion

15. Detection rate of TBTV, TVDV, TBTVsatRNA
and TVDVaRNA in feld collected samples.
RdRp nt sequences of TPV1, TPV2 as well as those of other viruses in the family Solemoviridae
Result and discussion

16. Summary
 This study found TVDV+TVDVaRNA+TBTV+TBTVsatRNA co-infection of different plant spp
(crofton weed, tobacco, stick tight, broad bean, pea, oilseed rape, pumpkin, tomato)
 This new finding indicates that the TBTD causal viruses, which is a large virus reservoir in nature.
 Many plants co-infected with these four causal viruses did not show typical TBTD-like symptoms.
 TBTD are a group of different viruses and have much broader host ranges
 NGS is an efficient and rapid technique for identifying and detecting novel viral populations

17. Thank you for your attention!!

18. TVDV Tobacco vein distorting virus
PeVYV-1 pepper vein yellows virus 1
PYLCV Pepper vein yellows virus 2
PLRV Potato leafroll virus
TV2 tobacco virus 2
TuYV turnip yellows virus
BrYV Brassica yellows virus
BWYV beet western yellows virus
BMYV beet mild yellowing virus
CABYV Cucurbit aphid-borne yellows virus
BChV beet chlorosis virus
MABYV Melon aphid-borne yellows virus
CtRLV Carrot red leaf virus
CpCSV Chickpea chlorotic stunt virus
CYDV-RPS Cereal yellow dwarf virus-RPS
MYDV-RMV Maize yellow dwarf virus-RMV
ScYLV sugarcane yellow leaf virus
CYDV-RPV Cereal yellow dwarf virus - RPV
SABYV suakwa aphid-borne yellows virus