Molecular analysis of the complete

genomic sequences of Tobacco

etch virus isolated from China

Julong Cheng

Tobacco Institute of Shaanxi Province

2013

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Molecular analysis and complete

genomic sequences 3

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Background 1

Host range and symptoms 2

Identification of TEV in five tobacco

planting regions of China 4

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Serious losses:

2001-2010, average annual epidemic

area of tobacco viruses were 11.8-25.6

ten thousand ha., which is about 11.8-

21.4% of total tobacco growing area.

Part 1 Serious losses

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Mix infection by

complex viruses

Many virus

species

Lack of effective

control methods

and reagents

Disease cycle

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TEV virion Inclusion body

Etching necrotic stripe Mesophyll necrosis

In 1985, TEV was found in tobacco and pepper in Shaanxi, China.

Discovery of TEV in China

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LIV:2 ～ 5d (20 ℃)

Virus particle: Flexuous, 723×11.5nm

Inclusion: wind wheel, column in cytoplasm

TIP:53℃

DEP:3×10-3

Nuclei acid : RNA，MW 3.16×106 Da

Transmission: Myzus persicae can transmit easily during 10 min in

non-persistent manner

First report of TEV from tobacco in China, and its

biological and physiological Characterizations

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serious losses

sustainable

development of

tobacco

agriculture

needs of

the tobacco

production safety

widespread

tobacco planting

area

Tobacco etch virus is a species of the largest

plant virus genus Potyvirus. TEV-Shaanxi

induced systemic symptoms including

mosaic and necrotic lines or etching in

Nicotiana tabacum and the efficiency of

TEV-Shaanxi transmitted by Myzus

persicae was higher than Aphis gossypii.

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Part 2.1 Host range

Nicotiana tabacum Nicotiana glutinosa

Datura stranonium

Capsicum annuum

Lycopersicon esculentum

Chenopodium amaranticolar Cucumis sativus

Lagenaria siceraria Vigna sinensis

Phaseolus vulgaris

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Part 2 Host range

Chenopodium amaranticolar

Nicotiana tabacum

Nicotiana glutinosa

Local necrosis

Systemically mosaic

and etching

Datura stranonium

Capsicum annuum

Lycopersicon esculentum

Mottling and

distortion

Cucumis sativus Lagenaria siceraria

Vigna sinensis Phaseolus vulgaris Not

Detected

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Aphid Vectors No. positive/No. inoculated Rate of transmission

Aphis gossypii 21/30 70%

Myzus persicae 26/30 86.7%

Table 2 The efficiency of transmission by different vector for tobacco etch virus

The aphid species and hosts for rearing were as follows: the green peach aphid,

Myzus persicae, on turnip (Brassica rapa L.) and the cotton aphid, Aphis

gossypii, on cucumber (Cucumis sativus L.). Using five aphids per plant, aphids

of both Aphis gossypii and Myzus persicae could transmit TEV-Shaanxi with high

efficiency of 70% (21 plants infected out of the 30 inoculated plants) and 86.7%

(26/30), respectively.

Aphid transmissibility 2013

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Molecular analysis and

Complete genomic sequences

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sample collection

Extraction of RNA

RT-PCR detection

clone

transformation

RT-PCR detection

Sequence Save

RACE

sequence

Sequence Splicing

sequence analysis

Technical route

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Part 3 .1 Total RNA extraction

Total RNA was extracted from leaf samples

infected with TEV-Shaanxi using the Universal

Plant Total RNA Extraction Kit (BioTeke, China).

cDNA was synthesized using Prime Script RT

reagent Kit (TaKaRa, Japan), according to the

manufacturer’s instructions.

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Primer Sequence (5’–3’) Location Amplicon size (bp)

TEV-F1 GCAATGGCGATGACCTA 8014-8030 1481

TEV-R1 TTCTCGCACTACATAGGA(T)15 9477-9494

TEV-F2 AGTGGGTTAGTGGTTGG 6845-6861 1605

TEV-R2 AAGAGTTTGTTCCGTGCT 8432-8449

TEV-F3 GAAGGTGACTCGGGTATT 5167-5184 1893

TEV-R3 GACTAACTGACTGGGACA 7042-7059

TEV-F4 AGCGTTTAGGAATCTACTG 4182-4200 1377

TEV-R4 AGATGCTTAGCCACTTCG 5541-5558

TEV-F5 ATCCTGGCAATAGTCTCC 2542-2559 1817

TEV-R5 TTGGCGTTGGCACCTGT 4342-4358

TEV-F6 TAAGGGTGAGTCGGGAG 1260-1276 1685

TEV-R6 GCCAAGTTAATTCGTCCC 2927-2944

TEV-F7 GCCTTACACACCACTCC 339-355 1196

TEV-R7 TATGCGTGCTTGCTTCTT 1516-1533

5’RACE PCR

TEV- Outer CAAGTTTGGGCATACACG 657-674

TEV- Inner CGCTGCTTCTTACGCTTTGG 607-626

Table 1 Primers used for TEV complete genome sequence amplification

Part 3.2 Primer designation

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Part 3 .3 PCR and RACE

• PCR were carried out using the DNA Engine Peltier

Thermal Cycler (Bio-Rad, Hercules, CA).

• RACE

3’ 5’

Approximately 1.5

kb was obtained

using primer TEV F1

5’RACE system

(TaKaRa, Japan).

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Part 3 .4 Cloning and sequencing

Purification

Amplified PCR products

pMD18-T

simple vector

Escherichia coli

JM109

Sequencing

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Fig. 1 Schematic representation of the genome organization of Tobacco etch virus

(TEV) and the putative protein cleavage sites compared to three other potyviruses,

Potato virus A (PVA), Tobacco vein mottling virus (TVMV) and Plum pox virus (PPV).

The number above each site indicates the first amino acid residue of each mature

protein in the TEV polyprotein

Genome structure of TEV

Part 3 .5 Genome structure of TEV

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TEV isolates can be clustered into four

genetic groups that are corresponding

with their geographical origin.

Part 3 .6 Phylogenetic analysis

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Phylogenetic analysis of the polyprotein

sequences of TEV and other species in

the genus Potyvirus imported from the

GenBank indicates that TEV is a

member of a subgroup.

Part 3 .6 Phylogenetic analysis

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TEV

isolates Geno

me Polypro

tein 5'-

UTR P1

HC-

Pro P3 6K1 CI 6K2

Nia-

VPg NIa-

Pro NIb CP

3'-

UTR

TEV-

DQ986288 95.6 98.0 93.1

95.0/9

4.7 96.4/9

9.6 96.2/9

7.4 98.7/1

00 95.6/9

8.7 96.2/9

8.1 96.5/9

7.3 96.0/9

7.9 95.9/9

7.9 97.2/9

8.9 80.0

TEV-

EF470242 99.6 99.4 100

99.6/9

9.0 100/

99.6 100/

100 99.4/9

8.1 99.5/9

9.1 98.1/9

6.2 100/

100 99.3/9

9.2 99.7/9

9.4 99.9/1

00 97.9

TEV-

NC_001555 96.2 97.8 94.4

95.0/9

4.7 99.8/9

9.1 96.5/9

7.1 98.7/1

00 95.6/9

8.1 96.2/9

8.1 96.6/9

7.9 96.0/9

7.5 96.0/9

7.9 97.3/9

9.2 98.9

TEV-

M15239 96.1 97.8 93.8

94.9/9

4.7 99.8/9

9.1 96.5/9

7.1 100/

100 95.6/9

8.1 96.2/9

8.1 96.6/9

7.9 96.0/9

7.5 96.0/9

7.9 97.3/9

9.2 100

TEV-

L38714 96.2 97.8 94.4

95.0/9

4.7 94.7/9

6.4 96.4/9

7.1 99.1/1

00 96.5/9

8.1 97.1/9

8.1 98.7/9

7.9 100/

97.5 95.6/9

7.9 98.1/9

9.2 96.2/9

8.9

Table 3 Percent nucleotide (left) and amino acid (right) sequence identities of

TEV-Shaanxi with other TEV isolates

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The complete genome sequence of TEV consists of 9,494

nucleotides, excluding the 3’poly (A) tail.

The genome contains a single large open reading frame

(ORF) encoding a polyprotein of 3,054 amino acids, with

an AUG start codon and a UGA stop codon.

The sequences of the separate genes (except the small 6K1

and 6K2) were compared with 32 other species of the

genus Potyvirus.

Summary

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Identification of TEV in five tobacco

areas of China

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Region infection rate (%)

Shaanxi 20.19

Yunnan 12.96

Hunan 12.39

Shandong 5.43

Heilongjiang 21.50

732 samples were detected and the results indicated:

TEV was evenly distributed;

Part 4 Identification of TEV in five tobacco

areas of China

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Summary In this study, we sequenced the complete genomic sequence

of TEV isolated from China and compared it with other

complete or partial (coat protein) TEV sequences available in

the sequence database. The purpose of this study was to

determine the complete genomic sequence of TEV for a

better understanding of the genomic organization, genetic

diversity and evolutionary relationship of the virus.

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Thanks

2013.10.15

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