CN106929520B - Tomato spotted wilf virus gene and application thereof - Google Patents

Abstract

The invention discloses a gene sequence (including RNA or DNA molecules) of a tomato spotted wilt virus, namely a hot pepper yellow mosaic virus and application thereof. The base sequence of the virus gene sequence is shown as SEQ ID NO:1, can encode nucleocapsid protein (N) and nonstructural protein (NSs) of a virus; or the base sequence of the virus gene sequence is shown as SEQ ID NO:2, can encode the movement protein (NSm) and glycoprotein (Gn or Gc) of the virus; or the base sequence of the virus gene sequence is shown as SEQ ID NO:3, capable of encoding a viral replicase (RdRp). The application of the virus gene sequence in preparing a detection kit for detecting the hot pepper yellow mosaic virus and the application of the virus gene sequence in preparing a medicament or a reagent for preventing and/or treating diseases caused by the hot pepper yellow mosaic virus.

Description

Tomato spotted wilf virus gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a tomato spotted wilt virus (namely a pepper yellow ringspot virus gene) and application thereof.

Background

The tomato spotted wilt virus belongs to the Bunyaviridae, and the virus particle of the family has spherical and enveloped virus particle and segmented negative strand RNA as genome. The tomato spotted wilt virus is named after spotted wilt disease caused by tomato infection, the diameter of the virus is 80-120 nanometers, a plurality of glycoprotein protrusions extend out of an envelope, 3 spirally symmetrical nucleocapsids are arranged in the virus, and the virus respectively contains 3 large (L), medium (M) and small (S) RNA segments, wherein L is negative strand RNA and respectively codes replicase (RNA dependent RNA polymerase, RdRP) and M is ambisense RNA of the virus and codes motor protein (NSm) and glycoprotein (Gn and Gc); s is an ambisense RNA encoding a Nucleoprotein (NP) and a non-structural protein (NSs). Based on serological and biochemical analysis, 8 and 22 viruses have been identified as possible members of this genus.

Tomato spotted wilt virus is the only genus of plant infection in the bunyaviridae family, transmitted by thrips, causing serious disease in economically important crops in tropical and subtropical regions. In recent years, the harm of tomato spotted wilt virus viruses is continuously reported in China, particularly Yunnan province, and serious loss is brought to agricultural production. However, the pathogeny of the capsicum yellow ring spot and the tomato yellow spot caused by infection is not clearly identified, and the disease is seriously prevented from being rapidly diagnosed, prevented and controlled in time. Therefore, the isolation and identification of the pepper yellow ringspot virus and the genome thereof are the prerequisite for the rapid diagnosis of the disease, and the solution of the above problems is very necessary.

Disclosure of Invention

The invention aims to provide a tomato spotted wilt virus (i.e. pepper yellow ring spot virus) gene; the second purpose is to provide the application of the tomato spotted wilt virus (namely, pepper yellow ringspot virus) gene.

The first purpose of the invention is realized by that the base sequence of the pepper yellow ringspot virus gene is shown as SEQ ID NO:1, can encode nucleocapsid protein (N) and nonstructural protein (NSs) of a virus; or the base sequence of the gene of the pepper yellow ringspot virus is shown as SEQ ID NO:2, can encode the movement protein (NSm) and glycoprotein (Gn or Gc) of the virus; or the base sequence of the gene of the pepper yellow ringspot virus is shown as SEQ ID NO:3, capable of encoding a viral replicase (RdRp).

The second purpose of the invention is realized by the application of the tomato spotted wilt virus gene in the preparation of primers, probes or kits for detecting the pepper yellow mosaic virus or antibodies for resisting the pepper yellow mosaic virus.

The application of the hot pepper yellow ringspot virus gene in preparing a detection kit for detecting the hot pepper yellow ringspot virus is provided.

The application of the capsicum yellow ringspot virus gene in preparing a medicament or a reagent for preventing and/or treating diseases caused by capsicum yellow ringspot virus.

Under the condition of unknown pathogeny, the inventor identifies and separates the disease by methods such as host range determination, virus separation, RT-PCR, serological detection and the like to complete virus genome sequence determination, the capsicum yellow ringspot virus is a virus which is firstly obtained by the inventor from infected capsicum and tomato, and the genome sequence information and functional genes of the virus are not reported at home and abroad. The acquisition of the virus genome sequence has important significance for understanding the virus source, the evolution process and the molecular epidemiology, lays a foundation for the differential diagnosis of the disease and the development of disease-resistant genes, and has great economic and social benefits.

The inventor utilizes the bioinformatics technology to input a keyword tomato spotted wilt virus (Tospovirus) from an NCBI website, obtains related nucleotide sequences through retrieval, designs degenerate primers for amplifying pepper yellow ringspot virus genomes according to comparison results through comparison, and utilizes the molecular biology technology to extract total RNA, RT-PCR, clone, sequencing and other technologies to obtain the whole genome sequence of the pepper yellow ringspot virus infecting pepper for the first time in the world. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. On the basis of this, the present invention has been completed.

Detailed Description

The present inventors have conducted extensive and intensive studies to obtain a pepper yellow ringspot virus whole genome sequence for the first time. On the basis of this, the present invention has been completed.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The complete genome sequence or a fragment thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the sequence. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In a first aspect, the invention provides a nucleic acid comprising a genome nucleotide sequence of a pepper yellow ringspot virus shown in SEQ ID NO1, SEQ ID NO 2, and SEQ ID NO 3. Also provided are nucleotide sequences comprising sequence identity to the nucleotide sequences disclosed herein. Depending on the particular sequence, the degree of sequence identity is preferably greater than 89% (e.g., 89.7%,90%,91%,93%,95%,99% or more). These sequences include, for example, mutants and allelic variants.

The invention also provides nucleic acids comprising one or more of the nucleotide sequence fragments disclosed herein. These fragments should comprise at least n contiguous nucleotides in the sequences, and depending on the particular sequence, n is 10 or higher (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 75, 100 or higher). Preferably, the fragment is unique to the genome of a tomato spotted wilt virus, in other words it is not present in the genome of other organisms. More preferably, the fragment is unique to the genome of a strain of tomato spotted wilt virus. The invention also provides nucleic acids that hybridize to the nucleic acids provided herein. Hybridization conditions are as described herein below.

The invention also provides a nucleic acid comprising a sequence complementary to the above sequence (e.g., for antisense, for a probe, or for an amplification primer).

Of course, the nucleic acids of the invention can be prepared in a variety of ways (e.g., chemically synthesized, prepared from a DNA library, or from the organism itself, etc.) and can take a variety of forms (e.g., single stranded, double stranded, vectors, probes, primers, etc.). The term "nucleic acid" includes DNA and RNA, and analogs thereof, such as those containing modified backbones, and also Peptide Nucleic Acids (PNA), and the like.

It is understood that since SEQ ID NO1, SEQ ID NO 2, SEQ ID NO 3 represent the complete genome of a tomato spotted wilt virus.

The invention also provides vectors (e.g., expression vectors, sequencing vectors, cloning vectors, etc.) comprising the nucleotide sequences of the invention and host cells transformed with these vectors.

In a second aspect, the invention also provides a protein comprising an amino acid sequence encoded in a nucleotide sequence of a hot pepper yellow mosaic virus as set out herein. Proteins comprising sequences identical to the sequences of these proteins are also provided. Depending on the particular sequence, the degree of sequence identity is preferably greater than 89% (e.g., 89.9%,90%,91%,93%,95%,99% or more). These homologous proteins include the mutants and allelic variants encoded in the nucleotide sequences of the pepper yellow ringspot virus as set forth herein.

The invention also provides a protein, which contains the amino acid sequence fragment coded in the nucleotide sequence of the hot pepper yellow ring spot virus disclosed in the sequence table. These fragments should comprise at least n consecutive amino acids in the sequences, and depending on the particular sequence, n is 7 or higher (e.g., 8, 10, 12, 14, 16, 18, 20 or higher). These fragments preferably comprise an epitope of the sequence.

The invention also provides nucleic acids encoding the proteins of the invention.

In a third aspect, the invention also provides a computer, computer memory, computer storage medium (e.g., floppy disk, hard disk, CD-ROM, etc.) and/or computer database containing the nucleotide sequence of the nucleic acid of the invention. Preferably, it contains a nucleotide sequence of a tomato spotted wilt virus as set out herein.

This can be used to analyze the nucleotide sequences of the pepper yellow ringspot virus listed herein. For example, a search can be performed to identify an Open Reading Frame (ORF) or coding sequence in a sequence.

In a fourth aspect, the present invention provides a method of identifying an amino acid sequence comprising the step of searching for a putative open reading frame or protein coding sequence within the nucleotide sequences of a pepper yellow ringspot virus as set out herein. Similarly, the invention provides the use of a nucleotide sequence of a pepper yellow ringspot virus as set out herein for searching for a putative open reading frame or protein coding sequence.

In a fifth aspect, the invention provides antibodies that bind these proteins. They may be polyclonal or monoclonal and may be prepared by any suitable method known to those skilled in the art.

The antibodies of the invention can be used in a variety of different ways, e.g., to confirm expression of a protein, or to confirm the location of expression of a protein.

The invention provides a nucleotide sequence of a capsicum yellow ringspot virus and an amino acid sequence coded by the nucleotide sequence. Using these disclosed sequences, nucleic acid probe assays and expression cassettes and vectors can be generated. Proteins may also be chemically synthesized, and expression vectors may be transferred into host cells to produce the proteins. The purified or isolated polypeptide can be used to generate antibodies for detecting pepper yellow ringspot virus proteins. Furthermore, the host cells or extracts can be used in biological assays to isolate agonists and antagonists. Furthermore, using these sequences, one can search to identify Open Reading Frames (ORFs) and to identify amino acid sequences.

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.

The base sequence of the tomato spotted wilf virus gene is shown as SEQ ID NO:1, can encode nucleocapsid protein (N) and nonstructural protein (NSs) of a virus; or the base sequence of the tomato spotted wilt virus gene is shown as SEQ ID NO:2, can encode the movement protein (NSm) and glycoprotein (Gn or Gc) of the virus; or the base sequence of the tomato spotted wilt virus gene is shown as SEQ ID NO:3, capable of encoding a viral replicase (RdRp).

As shown in SEQ ID NO:1 is shown as SEQ ID NO: 4. SEQ ID NO: 5, and a sequence or molecule having an amino acid sequence homology or similarity of greater than 89.9%; as shown in SEQ ID NO:2 is shown as SEQ ID NO: 6. SEQ ID NO:7, and a sequence or molecule having an amino acid sequence homology or similarity of greater than 89.9%; as shown in SEQ ID NO:3 is shown as SEQ ID NO: 8, and a sequence or molecule having an amino acid sequence homology or similarity of greater than 89.9%.

The separation method (or preparation method) of the gene sequence of the pepper yellow ringspot virus comprises the following steps:

1. collecting disease sample and propagating virus

The method comprises collecting tomato spotted wilt virus (also called pepper yellow ringspot virus), separating Kunuori single spot as a blight host, mechanically inoculating, and storing on the tobacco.

2. After the disease of the tobacco occurs, the symptomatic disease leaves are taken to extract the total RNA

Extracting total RNA of a disease sample, removing a disease plant, grinding by liquid nitrogen, extracting the total RNA of the disease sample by TRIzol purchased from Invitrogen, using 12 muL of total RNA as a template, mixing 1 muL of 100 muM downstream primers, quickly inserting the cDNA into ice for 5min after 10min of pre-denaturation at 70 ℃, adding 4 muL of 5 xRT Buffer, 2 muL of 10mmol/L dNTP, 1 muL of 5U/mu L M-MLV, 1 muL of 40U/muL RNase Inhibitor, and extending for 90min at 42 ℃. PCR amplification was performed using cDNA as a template or a vector. The reaction system is 50 muL: 3 muL of cDNA, 1 muL of an upstream primer, 1 muL of a downstream primer, 1 muL of dNTP, 1 muL of rTaq, 5 muL of 10 gamma buffer and H2O 38 muL. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min for 30s, and cyclic amplification for 30 times; and finally extension at 72 ℃ for 10 min.

3. And (3) cDNA synthesis:

the cDNA synthesis system is 20 muL, 12 muL total RNA is used as a template, 1 muL 100 muM downstream primers (table 1) are mixed, after pre-denaturation at 70 ℃ for 10min, the mixture is quickly inserted on ice for 5min, 5 xRT Buffer 4 muL, 10mmol/L dNTP2 muL, 5U/mu L M-MLV 1 muL, 40U/muL RNase Inhibitor 1 muL is added, and extension is carried out at 42 ℃ for 90 min.

TABLE 1 preparation of primers for a Hot Pepper yellow ringspot Virus

The application of the tomato spotted wilf virus gene is the application of preparing a primer, a probe or a kit for detecting the hot pepper yellow mosaic virus or preparing an antibody for resisting the hot pepper yellow mosaic virus.

The gene sequence of the hot pepper yellow ringspot virus is applied to the preparation of a detection kit for detecting the hot pepper yellow ringspot virus.

The gene sequence of the hot pepper yellow ring spot virus is applied to the preparation of medicines or reagents for preventing and/or treating diseases caused by hot pepper yellow mosaic virus.

The application of the gene of the hot pepper yellow ringspot virus is the application of the gene in preparing a detection kit for detecting the hot pepper yellow ringspot virus.

The length of the primer is 15-100 nucleotides.

The length of the probe is 25-5000 nucleotides.

The length of the probe is 50-500 nucleotides.

The application of the virus gene sequence in preparing the detection kit for detecting the hot pepper yellow mosaic virus comprises the following specific operation steps:

extracting total RNA of a disease sample, removing a disease plant, grinding by liquid nitrogen, extracting the total RNA of the disease sample, taking 12 muL of total RNA as a template, mixing 1 muL of 100 muM downstream primers, quickly inserting the DNA into ice for 5min after pre-denaturation at 70 ℃ for 10min, adding 4 muL of 5 xRT Buffer, 2 muL of 10mmol/L dNTP, 1 muL of 5U/mu L M-MLV, 1 muL of 40U/muL RNase Inhibitor, and extending for 90min at 42 ℃; using cDNA as template or PCR amplification; the reaction system is 50 muL: 3 muL of cDNA, 1 muL of an upstream primer, 1 muL of a downstream primer, 1 muL of dNTP, 1 muL of rTaq, 5 muL of 10 gamma buffer and H2O 38 muL; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min for 30s, and cyclic amplification for 30 times; finally, extending for 10min at 72 ℃;

recovering the PCR product by using a UNIQ-10 column type PCR purification kit or a UNIQ-10 column type glue recovery kit, wherein the recovered fragment can be used as a probe; meanwhile, the DNA can be connected to a pMD18-T vector, and the connection system is as follows: 2.5. mu.L of Ligation mix, 0.5. mu.L of pMD18-T, 2. mu.L of PCR-recovered product, ligated overnight at <16 ℃; the DNA was transformed into E.coli DH 5. alpha. by heat shock at 42 ℃ and positive clones were screened by colony PCR and sequenced.

The virus gene sequence of the separated DNA molecule is SEQ ID NO:1 or 150-3428 continuous nucleotides in the antisense sequence; SEQ ID NO:2 or 150-4781 continuous nucleotides in the antisense sequence; SEQ ID NO:3 or 150-8917 continuous nucleotides in the antisense sequence.

The sequence of the separated DNA molecule is SEQ ID NO:1 or 300-3248 continuous nucleotides in the antisense sequence; SEQ ID NO:2 or 300-4781 continuous nucleotides in the antisense sequence; SEQ ID NO:3 or 300-8917 continuous nucleotides in the antisense sequence.

The sequence of the separated DNA molecule is SEQ ID NO:1 or 1000-3248 continuous nucleotides in the antisense sequence; or SEQ ID NO:2 or the antisense sequence thereof, and the antisense sequence thereof consists of continuous 1000-4781 nucleotides; or SEQ ID NO:3 or the antisense sequence thereof, and the antisense sequence thereof consists of continuous 1000-8917 nucleotides.

The application of the DNA molecule in preparing the detection kit for detecting the hot pepper yellow mosaic virus comprises the following specific operation steps:

1. cDNA Synthesis

The cDNA synthesis system is 20 mu L, 12 mu L total RNA is used as a template, 1 mu L downstream primers of 100 mu M are mixed, the mixture is quickly inserted on ice for 5min after pre-denaturation is carried out for 10min at 70 ℃, 5 xRT Buffer 4 mu L,10 mmol/L dNTP2 mu L, 5U/mu L M-MLV 1 mu L and 40U/mu L RNase Inhibitor 1 mu L are added, and extension is carried out for 90min at 42 ℃.

2. PCR

Carrying out PCR amplification by taking the cDNA as a template, wherein the reaction system is 50 muL: 3 muL of cDNA, 1 muL of upstream primer, 1 muL of downstream primer, dNTP1 muL, rTaq 1 muL, 5 muL of 10 gamma buffer, H₂O38 muL. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min for 30s, and cyclic amplification for 30 times; and finally extension at 72 ℃ for 10 min.

3. Cloning and sequencing of the Gene of interest

And (3) recovering the PCR product by using a UNIQ-10 column PCR purification kit or a UNIQ-10 column gel recovery kit, connecting the recovered PCR product to a pMD18-T vector, and connecting the recovered PCR product to a system:

the DNA was transformed into E.coli DH 5. alpha. by heat shock at 42 ℃ and positive clones were selected by colony PCR and sequenced by sequencer.

The invention is further illustrated by the following specific examples:

example 1

Propagation of viruses

The pepper disease samples retrieved in the field were subjected to 2 single spot separations on Kunnuoli to obtain purified viral isolates which were preserved on Nicotiana benthamiana and Nicotiana annua for use in the extraction of total RNA from the disease samples.

RT-PCR

1g of pepper yellow ringspot virus infected Nicotiana benthamiana leaves are taken, TRizol is used for extracting total RNA of a disease sample, the total RNA is used as a template, and RT-PCR amplification is carried out, wherein reverse transcription primers are as follows: 5' -CCC GGA TCC AGA GCA ATNNNNNNN in 0.2mlSequentially adding the following components into a PCR tube: 5 × buffer 6 μ L, 3^，primer (50U) 1. mu.L, template RNA 10. mu.L, mix well, 70^oDenaturation of C for 10min, immediately placing on ice for 5min, adding 10mmol/L nNTPs 1 μ L, AMV-L1 μ L, RNase 1 μ L, 0.1M DTT 1 μ L, DEPC H₂Mixing O9 μ L to total volume of 30 μ L, and 42%^oC extends for 1 h.

And (3) PCR: a PCR reaction solution (50 μ L system) was prepared according to the following, including 5 μ L of 10 x buffer, 2 μ L of 10mmol/L dNTPs, 3 ', 5' -primer (5 '-CCCGGATCCAGAGCAATNNNNNNNN-3', 5 '-NNNNTTTTGTTTTTGTTTTTTGNNNN-3') each 1 μ L, 5 μ L cDNA, 1 μ L Taq, H-complement, and the like₂O35. mu.L. The reaction conditions are as follows: 94^oC Pre-denaturation for 2min, 52 ^oC annealing for 1min, 72 ^oC extension for 2min for 30 amplification cycles, preferably 72^oAnd C10 min. After the reaction, 3-5. mu.l of the reaction product was subjected to 0.7% agarose gel electrophoresis.

The target fragment is purified by tapping, connected to a pGEM-T vector, positive clones are screened by colony PCR with M13 primer, the obtained clones are sent to a sequencing company for sequencing, and the independent single clone containing the target fragment is subjected to bidirectional determination. Through repeated sequencing for many times, the complete sequence of the genome of the pepper yellow ringspot virus shown in SEQ ID NO1, 2 and 3 is obtained on the basis of average sequencing about 6 times per base, and is determined through sequence analysis and comparison.

Example 2

Verification of genome sequence of pepper yellow ringspot virus

According to the nucleotide sequences shown by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, primers are designed according to the length of about 1200bp per 800-. The results showed that the nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 were correct.

Carrying out PCR amplification by taking the cDNA as a template, wherein the reaction system is 50 muL: 3 muL of cDNA, 10 muM 1 muL of upstream primer, 10 muM 1 muL of downstream primer, dNTP2.5 muM 1 muL, rTaq 1 muL, 10 xbuffer 5 muL, H₂O38 muL. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min for 30s, and cyclic amplification for 30 times; most preferablyThen extending at 72 ℃ for 10 min.

The verification method comprises the following steps:

inputting the sequence obtained by sequencing under a blast page of an NCBI website to perform blast comparison so as to verify whether the sequence is correct.

Example 3

Detection kit for detecting pepper yellow ringspot virus

The nucleotide sequence disclosed by the invention is utilized to obtain target protein in a prokaryotic expression mode, obtain polyclonal antiserum by immunizing rabbits, and obtain monoclonal antibodies by immunizing murine tumor cells. These antibodies were used for detection as follows.

Double antibody sandwich ELISA (DAS-ELISA)

Coated capture antibody 100. mu.L of capture antibody diluted in coating buffer (coating buffer) was added to each well, containing wells corresponding to positive, negative and blank controls, and left at 37 ℃ for 3 h.

The antibody was spun off with coating antigen, washed 4 times with PBST, and patted dry. Add 100. mu.L of PBST-diluted plant disease-like crude sap per well, set positive, negative and blank controls, and stand at 37 ℃ for 3 h.

And (5) sealing, throwing away the coating antigen, washing the plate for 4 times, and standing for 3-5 min each time. mu.L of blocking solution (5% skim milk or 1% bovine serum albumin) was added to each well and left at 37 ℃ for 30 min.

The blocking solution was spun off with the enzyme-labeled antibody, the plate was blotted dry, 100. mu.L of enzyme-labeled antibody diluted with PBST was added to each well, and the mixture was allowed to stand at 37 ℃ for 3 hours.

The enzyme-labeled antibody is thrown off after color development, the plate is washed for 4 times, and a substrate (p-nitrophenylphosphate) is dissolved in 10 percent of substrate buffer solution, and the solution is developed for 15 to 30min at room temperature with 100 mu L/hole. OD was read at 405 nm.

Taking a pepper yellow ringspot virus infection sample, grinding the sample, diluting to 10, 50 and 100 times, and detecting by using the method, wherein the result shows that the obtained antibody can positively react with the sample infected with the pepper yellow ringspot virus.

Example 4

Detection kit for detecting pepper yellow ringspot virus

Based on the genomic sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, the following PCR primers and probes were synthesized:

a sense primer: the sequence is 1300-1320 of SEQ ID NO. 1.

Antisense primer: the sequence is the complementary sequence of the 2400 st-2420 th position in SEQ ID NO. 1

And (3) probe: the sequence is the 1786-2000 th position in SEQ ID NO. 1.

A kit (50 assays) was prepared containing: 50. mu.L of sense primer at a concentration of 100pmol, 50. mu.L of antisense primer at a concentration of 100pmol, 50. mu.L of probe at a concentration of 100pmol, and 2.5ml of RT-PCR reaction solution.

The method comprises the steps of taking a pepper yellowing ringspot virus infection sample, grinding the sample, diluting the sample to 10, 50 and 100 times, and detecting by using the method, wherein the result shows that the probe can positively react with the sample infected with the pepper yellowing ringspot virus.

Example 5

Based on the genomic sequence shown in SEQ ID No. 2, the following PCR primers and probes were synthesized:

a sense primer: the sequence is 1320-1340 in SEQ ID No. 2.

Antisense primer: the sequence is the complementary sequence of SEQ ID No. 2 at 2700 th and 2720 th positions

And (3) probe: the sequence is the sequence of position 1986-2200 in SEQ ID No. 2.

A kit (50 assays) was prepared containing: 50. mu.L of sense primer at a concentration of 100pmol, 50. mu.L of antisense primer at a concentration of 100pmol, 50. mu.L of probe at a concentration of 100pmol, and 2.5ml of RT-PCR reaction solution.

The method comprises the steps of taking a pepper yellowing ringspot virus infection sample, grinding the sample, diluting the sample to 10, 50 and 100 times, and detecting by using the method, wherein the result shows that the probe can positively react with the sample infected with the pepper yellowing ringspot virus.

Example 6

Based on the genomic sequence shown in SEQ ID No. 3, the following PCR primers and probes were synthesized:

a sense primer: the sequence is No. 3 at position 1720-1740.

Antisense primer: the sequence is the complementary sequence at position 3100-3120 in SEQ ID No. 3

And (3) probe: the sequence is the sequence of position 1986-2200 in SEQ ID No. 3.

A kit (50 assays) was prepared containing: 50. mu.L of sense primer at a concentration of 100pmol, 50. mu.L of antisense primer at a concentration of 100pmol, 50. mu.L of probe at a concentration of 100pmol, and 2.5ml of RT-PCR reaction solution.

The method comprises the steps of taking a pepper yellowing ringspot virus infection sample, grinding the sample, diluting the sample to 10, 50 and 100 times, and detecting by using the method, wherein the result shows that the probe can positively react with the sample infected with the pepper yellowing ringspot virus.

SEQUENCE LISTING

<110> institute of biotechnology and germplasm resources of academy of agricultural sciences of Yunnan province

<120> tomato spotted wilf virus gene sequence and application thereof

<130> 2017

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 3428

<212> DNA

<213> base sequence of tomato spotted wilt virus gene

<400> 1

agagctatcg gggcttctaa taattctaga aaaacaacga tataatcaag aaatactatt 60

tcaggcatgt ctactgcaaa gagtgctgct tctgaattca tcaaaagcta tggcacaaga 120

gacaatcgag caatcaatga ttgctactct gtcttctctg gagaaggtgt caactttctc 180

aatttgttta tgcataacaa tgcaggtatt aaatctgcat tcagcatcaa cgatttgggc 240

aggaatgagg atgtcaaaat ccatgaagct gaggttattg attcatgcca tgattatcat 300

tattttgaga aatttgggtt agatataaca ttttgcgaac atgaaatgaa cttgatcgtg 360

agaaagcctg gcataaagaa cacaggctgc aagttctcca tgcatagcca aattttcaac 420

ccaaatccag atatgttggc tttaattcct ggaacagttt cagaggagga tttttacgag 480

aagagcaaaa taaggattga tgggttactt ccttcaggat ggtgcttaga tgaatgctgg 540

aaaaataatt tctacatagc cactaacggg gacttcaatt tagattatgg tttctctgtc 600

atgggcaaaa caacatctta ttggagagaa agcattccaa aggaaaagat tctgtctgtg 660

aaacaaaagt gcttgcctga taacactgtc cccaccaacc gactgttatc cacatcaaca 720

gtcaaaggta ttcagcttgg ttctgaattg gcacatgata ccacagtgat actgtcatcc 780

aagcaaaatt tgaatactga tttgaaatct caatatcgca tttcatttca tggtatccaa 840

gaagagggtg ctttttcaag gactttctgc attccttttg aaaacaaatc cagaatgatt 900

tgtttatatg caaagaccgt tgctgacaac agcaatgaga gaaccacctt aattattaag 960

gtggttacaa aaactgtcga ctctcatctg gttatcccta tcagaaacca tatcaattgt 1020

gggaggaaag ttggagccag aataggtctt gttgatttcc ttgaaagtga ccctaattat 1080

aatcaaatga ttgttaagga gttgttgact gtgcacactc agtttgctat taacctatct 1140

gaggttatga agagaccaat tattgtgttt aaaatgtatg acagggagtt gcgcaatggt 1200

catactgaag tgcttgggag agtcttgaac taccaaatag atgatgaagg gaacatttat 1260

ttcctttcaa aaactcttga agttcttcca aaatcacttt ccactctaag ttatttaggg 1320

agtatcgcac cagttcagtg gaaagaatct ctggaacacc agcatttcgc tgtaaaagct 1380

gaataaacct atatgaagtt gtcattctat ttcagtatta ttgcatgcat atattatata 1440

ataagtaaat atagctgtat ctgtttgaat aaataaattt aatctttagt ttatgtgtgt 1500

taggctttta agtgtctctg tcacaatttc attactgtaa gtttgtctat ataaataaga 1560

tagatattat catatctcga gtttatgtgt gttaaacatt tgaatatctc tgttacaatt 1620

tcatcaatgc aaccttatct atataattaa gataaatatt attatattta agatatataa 1680

ttttaagtca taaataagct aagaaaaccc ataaaaatca aataaacaaa aaaccaaaaa 1740

aaagtcctcg aaagaggcaa aattggcatt tctaatttag gcagtttaat tcaatcaaaa 1800

taaatcatta cttaatttat tgacattatt tcataatata tgaaatgaat gcaatttaat 1860

ttttgtcagt cacacttgaa ttcctcttgg ttcgaatatc aagtttttaa tgttggatag 1920

attcagttaa ttatcaagca attatctctt ttgaataaac accatgaaac tcatccaaat 1980

ttaaatgcaa atttattaag taaccttaat cattcatgaa ttgcaattta tgaacatatt 2040

aatcagttca agttatatag gtttaatttt aatttctgaa atagattgct ttctttattc 2100

agttgtatgg ttatatattc agcatatcaa gaatgtaaca aattcaggga gtctttgaat 2160

cctataaatc agtttatttg aatcaatagg tctttataat ttattagatc tatatttgca 2220

gaaatgcggc caccgattgc ctccgaagag gacttttttt ggttttttga ttttttgatt 2280

tttttgtgtt tttatttgtt ttcaacttaa cttaattatt tatttattta tttatttatt 2340

atatttaaat tattttactt aacatacata aacagaaata taacaagcaa actgtatatt 2400

ttaattatat atataataat aaaatatata aacaaactta tacgcacaca caaacattta 2460

aaaaatattt atagaattgt aaacacacat aggtacattg gtgtacacat atatatcttt 2520

aatactgttt gaatcacact tccagagaag aacttggttt tgagcttttc tttgaatgtt 2580

caccataatc atccacagta aggctagcac tgaaaactct gtccatatat ttaacttgat 2640

catcatattt cttgagtgag ttagaactgg ctgtcccagg attgctttca ctgagcattt 2700

tgacagctgc tttaaacaag ctatccaaat catccttaaa ctcaatctgt gaagcagaaa 2760

ggaccttagc cactttgcaa acctgttcat aagtagagaa attctttatg ccaagttttt 2820

cttttttcac attttgataa taagatagag gaaatatgat cggtgcaagg cccttgatac 2880

tggacaaaag aggtagagga cctccaatgc acagcatcat ccttaaagca caggaatcaa 2940

aagatgcagg tacattcaat ccgtatgctg ctaccagtgg taattccatt atttttgcat 3000

acatatcctg tttagcagct tcatttttgt ttttctctac catgctgacc atttttgtcc 3060

taataaaagc ttctgttctt ttaaaggtcc aatcttctgg tccaacatca gctgatgtag 3120

ccacaatggt tttaccgcaa aaaacatact tgccattctt gcaggcagca aaaatctgct 3180

ttctgcattt taggatattc aaacagttca tgaaagttat ctcaatattc ttgttattct 3240

cataaaaagc tttgaagcta aatccaggag ttgagtcttc agtttcaatt tcaacatctg 3300

cagtaccgcc agccaaaaga tctttgattt tcttctctgt aagttgctta acgttagaca 3360

tggtgtttac tcttgaaacg tctagtatgt tgactttgaa atgattttat taacgcctcg 3420

attgctct 3428

<210> 2

<211> 4781

<212> DNA

<213> base sequence of tomato spotted wilt virus gene

<400> 2

agagcaatcg gtgcaccaat tactagataa atcatcgaat aatttccaag aaataatgtc 60

tcgcttctct aacgtgttag aatctttacg tcctgtaaaa agttcgaaca aagaattggt 120

acctgctgtc aagacagaaa ataacagaaa cattttggca aggaatgttt caagaaaaga 180

tatcgatgat gctatcatga acaaagcaaa gacaataaat gggaaacagt atgtttctgg 240

gggagattct agtgttttag ggacatattc cagtgaatta gctgttgaag ctacgtcgga 300

tgatattttg tctaggcttg ttgttgaaca aagcacccat ctgagcagct ggaaaaacga 360

ttcgcttgtt ggtaatggaa atgaaaaggt cagtttcact ataaatataa tgcctacatg 420

gaatagtggg cggagattta tgcatatatc taggcttata gtctggattg tcccaaccat 480

tcctgatgca aaaaataata ttaaagctac cttggttgat cccaacaaaa tgactcgtga 540

tgaaaagata gttatcagcc gacaatcttc actaaaagat cccatgtgtt ttatttttca 600

tttgaattgg tcatttccta aagagaggaa cactcctaaa cagtgtatgc agcttaattt 660

gacaagtgat gaaaagtatg ctaaaggagt tagctttgct tcagtcatgt attcttgggt 720

caagaacttt tgtgatactc ctattgctgc agagagcaac acatgtgatg tggtgccaat 780

caatagagct aaagtcattc aatctgctgc tttgattgaa gcatgcaaat taatgatacc 840

taaaggaact ggtgggaaac aaatagctaa tcaaattaag agtctgcaaa aagctgcaga 900

aaagttggca ttggaggctg aaaatgaaga tggcaatgct gatttagaga ttgagatgga 960

taattctatt gacatctgac aaaattgtgc tatccattta atctatgttt gaattgttta 1020

agtgtttgtt gtgtttatct attgagtata agtcatgttt aggtttatat attatcatta 1080

tactaagtgt gtttaaatta aatatttgtg tgttatgttt aaaaatatgt gtatgtgcat 1140

atatatatat agaaagccaa aaaagaataa aaataaaata aagtaaacaa aacagaataa 1200

acaaacaaaa caacaaaaca aaacaaaaaa caaaaacaaa aacaaaaaga aaaatatcat 1260

ataattaaat aaggccaagg ccaactttgg cttttcagcc tttttttgga ttttgttgtt 1320

ttatgttgtt tttaggttta taatgttatt tatctatgtt aatatgaatt aaatgtctaa 1380

agaaaagtca taaccctttg gaggtgatct tctcttcctg gtttgatctg attctgcaaa 1440

actggttaaa agaacacttg attctaatga ttccacaacg tcctctaact tttgtctcct 1500

tttatcaaca taatatgttc tggacaattt gaaaatgctg ctcacaatat acaaacctac 1560

acatgcagcc gcaatgacta agattattct aactaaatca aagaagttcc caaagaaaga 1620

agctatccaa ttgaatggtg ctttgatcca gtcccacaga ctagaaatag aagtgtcaga 1680

attatgtgta ttctcatcat gagcactttt gtcatcaaaa tgtattattg tatcttggtc 1740

tacttgagta aattcatcta cctgtatatc aacagtgagt tcctcttgat cttcaggaat 1800

caattttaaa gatttgtctg caatttcacc agaacaatag gcttttatag atttctcatt 1860

tggacctaag aatgtcccta tttggtcaga tttgaaagag caagtgtcca tcatcaatct 1920

tgatgagaac gttgtgtctg atgtgtaagt tatattgcaa ttgatacctg cagcacattg 1980

agaacatcca gagcagacca ttctaacttc tgaaagaata ggttttgttg gaatcttttt 2040

aaacatttcc ttgggcatgt ctatgatcat ttttaatttc ccaaccagga aatctttctc 2100

catatacagt ttattgttgt tttcatcaag atatgacaag tcttttgaag gagataaaac 2160

ataaatgttg ctatatgtat agagtccaca ctgcttgatg ttaacttttt tgtctccaac 2220

agcactacaa ctccatgtaa aatcattttg agataaagtt gcaggaactg ataaagggat 2280

tccatccatt gtaatttgag ggtgaccaaa tgatgatcct gaaaagtccc ctaaatcagc 2340

aatgtttcct gtgagtattt tttgctgttt agtcacagca aatagcttat cagtactcat 2400

gtaatcatta tgtaagtcaa ttgacatatc cagctgataa taatctgttt ggatagggga 2460

tctatctgag tgttttttac atgaataccc atctagtgat tttacgcaaa cttcagctgc 2520

aacatggctt tcaacaacct gatacacatt aactaaactt gagaggtcat aaatgtttgt 2580

acagtgccca caaatagacc cttcatttat agctaaacat ccaagttctt cacaacccca 2640

ccatgaagta ggtgtaacac agaaatctaa tacccccact ttaggtttct gcttcacgca 2700

ttctgtacag ctgcctgtgc atgttaccaa ataatcagct actgttgtgt caatttttgc 2760

agtagaatat ttgtatctta tatcatactc tacacctaca cttttaatgt agatcataaa 2820

ttccatagga gaatgtgcag catcgtcatt gagtaagtag atcgatcctg tgttagtctt 2880

tatatccatc tccaaaagat atctgtattt cccgtcaact tcagttgaaa acaccagaga 2940

ctgccttggc atcagatttt caggtggtat gtcgtctact ttcaagcttt tgtaaaattt 3000

atattcccga gctgattctt cctctagaat tttagattca gtagggctgt ccatgtatcc 3060

ttctctcaat ctgttggcta ttttaagact agttagatta cctttaaaag attgtccttt 3120

gtaatatttg ttaaatttgg tatcaggtat attttttata gatcttaagg aattgcagcc 3180

atttctgcat gcaaacaagt tttgagcttg attgttactt accatgcata agtctgaacc 3240

taaaatacag tcatttagtg atgttgctct agacatgggc acaccagatc tatatactgt 3300

ttcagtaatg acatttccta tagagcactc acatgtctca taaccattgt cagttattcc 3360

aaacttgtct gttgtcaaag aatctaaatt taagttgtaa tagcattttt ctacacataa 3420

gttaggctgt tttaatgcta tactcgatgg tatgtaagag ataagaattg atgctagaat 3480

cattttagtc aaaaaaacta aaaagttagt acttagtttt gtatttatag tgaactgaaa 3540

ttgctgaatc aaggttaact ttttccattc tgctttgtct ttcgtaaaaa gataacattc 3600

actagtgtga ctcttagatg ctttatcttg gttacaaaca cattcttcag agcatgaatg 3660

agttagaaat gagaaacatc cacagactcg gcattttaat gggaaatatg gccacaacca 3720

attaatggtc catagtatcg ggtatgttaa aatgccaatt atatcgtacc agatgctcag 3780

tgcatctttg gttttccaga tcagccatga aatagggaat gcaaccatta ggaagacaaa 3840

aatccatttg aagtaagaga aattcgcgca gaagaaaatc tttttaggct ctcctgagta 3900

tttcgaaaca cagtttctta caggtatgtc cactttggca attagactag atttgtcacc 3960

acaaagcaag aaatgattac catcaagctc ttcaggtttg aaatttattg tagaatcgcc 4020

tacgattggt ttaacatttt caggtttgga atttttaatg tttcttatca acactccaac 4080

actttcttct gatctcaact tgatttgata aggagatgac attgatatct ggtttatttt 4140

gcagtcacca gacaacctca cagaaagtcg tgcgacagta ggagatttta ggtttattct 4200

agggtcgatg ttgattgggt tacttgattt gtcaacaaag aaaaattttg taccaatctc 4260

taaaactttt ttattttcca actttaaaac aggaactact ggtaaatctt tgaatttcat 4320

cttcttgaat tctttctcat attgacaaat gtcaaatatg ttagcagagc tgcttatgca 4380

cgacacaatt tccttaccac tgtcaatctg ataatgagca ttgaaatcac ttacaccctt 4440

tatcatgcag tatctttttt caaagttgtc acagcttaat tcagagattg ttgactgcgt 4500

tgttgtttca tcatccctca agatcctgga taatttcttt tgctttggtg gagatgtctc 4560

agttatataa acttcgtctt cttcgataag gtcatctggg tcatctacct tgtacctgtc 4620

ctgaatcttt ctcaactgga cactgttatc tgcctgattc aagaggtaga cctcagagac 4680

gaaaaagagc gaaagtaaac ctagacagta gacaagtaga tagtatttct tcatctttat 4740

gaggtatttc aagaatattt aattgttgca ctgattgctc t 4781

<210> 3

<211> 8917

<212> DNA

<213> base sequence of tomato spotted wilt virus gene

<400> 3

agagcaatcg aggaacaata agaatatcag taatgaacat tcaaactata aacagtttcc 60

tagatacttc aggcgaaatc accgtgtatc tccagacaat aaaagatgaa atcatgtcaa 120

tgtcaaacat cgtggatagg agctatcctg aaggcaaagg agtgattgaa tcaggcctaa 180

attccataat gtcaatgaat tacatcttag ataagattga agacatgaaa tctagatacc 240

atcaaagttc ttcttccatt gaccagaaag aaatggaaac agagattttt ctctttctag 300

ataaatacaa agagttggaa ttgatgaggc atgacctttt tggtgttcta gctggaagaa 360

ggctccattt tgctcctaag cataggtctg atgtcttcct gaaagattgt ctcatgtcct 420

acatagagtt ttgtaattca tctacatcta ttgtgaatcc aatagttgat attgatgact 480

taaaggaaaa gttagtcttc cagcacctta ctccagacaa ttacactata tacaaagaaa 540

ctaaaggtga aaaggcatgc ctgatgatat atgactggaa agtctccata gatgccatgt 600

ctgaaactaa gacctctgaa aattattaca caagtgtgtg gaagacattc aaagacatca 660

caattgatga acagcctttc ttagaaagac atcccatatt cataaccatt gttatattga 720

atccgagtgg gaacatgcca attgtggcca caactagcag agtcattcaa gagttcagaa 780

attcacctta tagtaatttc aatgatcgaa agaaagcagc cactgcagca aagttgatat 840

ctgtcaactc tctagggcag cttataggtt tgggagctga ttgctttcgc cgattttatt 900

cagagacaca agtattcaaa aactcacttc tggctaaggt tggtgagtat atgaatagat 960

cgtcagaagt tttctttagt cattggtcat atgagtacag gaaaaccaat ttatcgaaca 1020

atcaaattag ccaggacatt gttgatttaa ttgatgccat gcccaatgat gcaataaaga 1080

aagatatgct tgtccatttc ttatttggca gttacttata tttcaaagaa acaatgagtg 1140

atcttcatct caaagatagg tttgaagggt ataaagcaca ttgcgaaatg atggggatag 1200

aacctattga aacagaagaa aaacttaagg aatatcttga caaaaacgaa catctgtttg 1260

aaaacctgta tgaaggccat ttgggaaaaa taaaatccga tgttttgttc aggaaatcta 1320

aagaaataga gatagaaagt attgagagtg ctttcaacat aaatgcagaa gaataccaaa 1380

gggaatatcc tggatgtttc acaaatgatc ttcaagaaac caaaacaaat ttctctatct 1440

gttggtcccc aatgacgcaa gagttggaaa tgaatgaacg aaactataat aatgcaataa 1500

tacaatcatt tagagacact tttaccgatc aagtgaaact tatccatcac aagtcatatg 1560

gaggtagcag aatggatact catttctcaa taaccctttt taacctagta aaagcctgtc 1620

tagtagactt gagtacagac acaacaggtc actcaaaagc agctctggaa gatgttgttg 1680

atattaaaga tggaagtatc caggttaagc gaactgaaac ctctcaaaag tggaaagata 1740

taggaaaagt aaaaacaagg aatggtaatg agtttacaat atcatctgca tctgataatg 1800

aagctagaaa aaatttcttt aaaggtttga gtttgatgta tgttgacatg ggaaaaaaga 1860

aaaaaattga tataaaaaat gacctgaaaa gtaagattca agaatctatg gctgaaaaga 1920

aagatgaaga aataagcggt cctagtggag aatatgatgt atccaaagct gatatcccct 1980

ttcaaataag catcaaagga atcacacata agaaaaaact catcagacat gacaaccctg 2040

atgtaaagta ccattgtgaa tcaatgattg aagcaatgta tgttctctat ggcatggatg 2100

aaaggaaaga taaaaatagc aaaataaatt ctgtttacaa cgaatactgt tccaatcctt 2160

catcactttt tgctaagggt aatttgatag agacagagat gatcattgct aaaaatatcc 2220

atgaagtttc taaagaactt tccgtatata cgtatagtga agacatgatg cagatggcaa 2280

agggattaat ggtagctgat aggttcatga agaaaacaga ttttaaaatc ttgacctgtg 2340

ctaatacaag catgttatgt ctagcattca aaggagatgg tataaacaca gggaaatcag 2400

gagtcccata tataactttg catagagttg aaaaagatat acaaccttat tttgcttctt 2460

tatacacaaa agagcttatc gtctcattca aatcagataa ttatttcatc aacattatga 2520

gaccacagag acttaaccaa gttaggctct taagtttgtt taaagctccc agtaaagttc 2580

ctgtactttt ctcacaatat tctttgctga gcacagagat aaaaaaatgg ttgaatcaat 2640

cttctgtgga cgttttcaca tgccctgaaa acaaaatcca atatctccaa aagatattat 2700

tttcttctgt gatcataggg actgttacca agcttagcag aatgggaatt tttgatttca 2760

tgagatatgc agggttctta ccattgtctg attattcaaa tataaaagaa tatatagctg 2820

agaaatttga tcctgatatt acaaatgtta tagattgttt ctttgtttca ggtataaaaa 2880

atcttcttct gaaaatggaa gggatcaact tgagcaacag cataaagcct ttgaccatag 2940

accaagaaaa tgatatgtca ggaggaatat cagatttaga tattgtttgt cctattacag 3000

ggtctacact caagaccata gaatgcttat acaacaatgt ctaccttgca atctacatga 3060

tgcctaaatc tcttcacacc catgtgcata atctcaccac acttttaagt gttcctgctg 3120

aatgggagat caaattcaga gaaaaaatgg gatttttaat aggcgatgaa attaagccaa 3180

agaaagaaat gtttaatgat tctggaccat tctctatcaa tggagtgctt aatgtaaaaa 3240

ctttatttga ctattataaa aagaacatta tcaatgttgg tgcttgtaga tcaaatatag 3300

aagacaaaga ggatttttta tctgcccctt ataagataaa aactctaact tcttcaaaga 3360

agtgctcaaa agcagatatc atcaagaatt ctgaaatagt tgcagcttta aaatcaacat 3420

ttgggaaaca gcctgatgat attaaaggat ctgatctcta catattgaaa ggagttttga 3480

aatgttttga agaagatagg gaatcattaa taaacttcct ggaaattgaa agcttagatg 3540

aatcaaaata cctgcatttc ttttctaaca tgatgtcagg aaacaacaag gtgttgatga 3600

aaacaaatca agacaaattt tattacaaaa gtcatcctct tacagttgaa acattcatga 3660

aagttagata tggttatttt aataccacta cagttttaac gtcaaaaaaa gtaagtgaag 3720

aactatatga tctaatcaag gagtttaata agataacaga aatagatcta gaagctttgg 3780

aaaagctagg cagaggattg aaaggcaaca gagtgacatt tatacagcta ctagaattca 3840

ttttaatgaa atctaggact aatgctggta acacagattt ccttgtatca gtgtttgaga 3900

aaatgcagag aacaaaaatg gacagagaaa tttatcttat gagcatgaaa acaaaaatga 3960

tgttgtattt cattgaacat acttataaac atgtagctca atctgatccc tctgaggcaa 4020

tatcgatatc tggagattac aagataaaaa atcttgcatc tctgtcatat gacactataa 4080

ctaactataa tacagcactt cagaaaaatc ttgaatgcaa aatggctttc ttatcagctg 4140

atcagtcaaa atggtcagca tcagatttaa cctacaaata catactggct gttatcatga 4200

accctatcct gactacaggg gaaataaatt taatgtgcga atgcataatg atgtatgtta 4260

aattgaagag ggtatgtatt cctactgata ttttccttaa cttgaaaaga ggtcaaacac 4320

agtatggttc ttatgggaca gctttatcta tgttaacaga taatttagaa acaaacacat 4380

tcccggtatc tatgaattgg ctccaaggaa atctaaatta tctatcatct gtgtaccatt 4440

cttgtgccat gatgggttac gagaaagcca tgaaaaaaat gaaggattat gactttacaa 4500

taagatggat ggtgcattct gatgataatg ctacatctat ggtagttaga ggagatatta 4560

aaaaacttct ttccagtttt aattgctcta gtttatcaga actattgttt cgaagtatcc 4620

agtcgcattt taagagttat tgcataacgt tgaaccctaa gaaaagctat gcttcagaat 4680

cagaagtgga atttatatca gaaagaatta tcaatggtgc tgtaatacct ttatactgca 4740

gacatttagc taactgtagc actgaaagtt ctcacaacag ttattttgat gatttaatgt 4800

ctctttctat tcatattaca atgcttttaa gaaaaggctg tcctaatgag ttgataccat 4860

ttgcctatgc agcaatacag acccagtcac tcagcatata ttcaatgctg cctggagaag 4920

aaaatgatat atcttctatt attaagggaa ccaatttccc tctatcaaag agggaaatac 4980

ccatttgtgc tggaggttgg atgtatgctc ccattgaact gctttctatt ctgggacctt 5040

catcaaatga tcaattgatc tattataaaa taatattaga tttttttgac ctgagagatt 5100

ttaatagttt aaagaaaagt gttaactctc taggttatat tgatttgagg attaatgaat 5160

tgttcaaaag aataaaatat cagaaaacga catgtgctga tagaaagatg atatgcatgg 5220

taaatttatt taagacaagc ttgatgtctg aagattgcga taacttaaac gtaggaatga 5280

aatttcagtc aatgataaca cagatcataa aattacccag ttttgtcagt gagggttcat 5340

tacttaaaaa ctcaagtttc caagatttct gcaaaccgtt tccaaactta aaaaagaata 5400

cagatatact tgatgcattg aaacctagca acttaaatga aaaagattta gaagaggtaa 5460

gtgaatctaa gtttttatct caaatccaat tggaagaact gagcagacat atgtcatgtc 5520

atccagagtc atttttgatt gcaccaatga atgataaaga ttatatttta acaaatcttt 5580

acacatacag cagtgtaagt aaaagaaacc aaatgtcaaa tcaatctact gaaaaattgg 5640

cattggatag aattttaaga tcaaaagcta aaacattttt agatccgaat tcaaaagaga 5700

tggtttctta taaagacaac atgtcaatga agatgacaga aataatgaat cctactggaa 5760

atgatttcaa aataataaac acaatatctg gattgatggt aagagacatg aattttgaga 5820

tggttgtttc tttgatggaa aacacagttg ctaatgcttc cattcctaaa gctaattata 5880

atttcagatg gttcattaca gagaaagtcc ctagtgtgat tgaaggatca cctggtttaa 5940

ttgtaatgtc agcagtctat ggtatggatt atttaataga attagggtta aagaaactgc 6000

ctctgacaga gatgtctatc tcaatattgc atgacatctt tggaaatagg aaaacatttg 6060

atgatgttaa agaatgtatt caaaaaacca acaaccaata caagacagaa gagtttcaaa 6120

ataccaatga tctgaaaaga tatgtcttga gtattaatta catgattcaa tctcagaaca 6180

aactgctatc cattaacaca tgtttttcta gaaaaaactt cccattttat tccaagtaca 6240

atttgggcag aacatttatc actaacaccc tggccatatg gagcacaata tatagccgat 6300

tgacaaatat aaacttctat actaatttga attttgttat tgatagaagc tctagactta 6360

ttatatcgtt acagcgtgat atgaatctgg agaaattaat tgattgttgt gcatatgttt 6420

ctgataggct gcaaagtttg tttcctgata tgaccataga ctccattaaa cagattttac 6480

atagattaaa cttcaatgga gtagatctca tgaagaagat gaaatcagaa attacagaag 6540

tgaaaagagc tataaataac ataaaaacat ctactcatgt cactctttcc ttcagaccac 6600

agatgattgc aatgagcaaa catgcagcat ggctatataa ttttgggtat ataaatgaaa 6660

aagaatttaa atttgtgata gatcaaatca ggcaaagtga agtacattac atcaagacag 6720

atgaacaaga tgttaggggt tattatgttt caggtcatac atacaagatt ggaattaaaa 6780

ctcaatacaa ttatggacag ctagccatgt caaaccaaga tatatcaatc catctgcatt 6840

caccttatga ataccagaga gaagatgatg ggaaaatatg ggaagtccat gttagatctg 6900

cctacaagct ccttcagaaa ctgctgatag acaagcaaag tgctattaag acgtttctca 6960

atatcagaac cgaattaatg cctaatcagt tttgcattca tgaaagctca aataaaacac 7020

ttctaatatt gattaatgat actacgaaac caattgcttt ggaaagaatc aaattcaaag 7080

gcgatataaa atacatacct tcctctgatt tcgcttggaa catgatgagc aaccaatcaa 7140

aatataagct gagaccggct gaaactggag aatgttatac tgaattatat aaaatgatag 7200

acagcaatga tggactaatg aataccatcc taagcaattt gaaaaaaagc ctggaatatg 7260

gcaatgagat ggaaaactta atagagcaat caataaaaga cattgatgat gaagaaacca 7320

ttgattacat tagggattca gttgatcaga tccacaaatt ggctgttgaa ggtttggaaa 7380

caagcaaaac atcagaagag ttcgagaatt atctaaaaag agttgatttt caatccactg 7440

tcgatttcca taaagatttg ttggagcaga ttgttgaaga aaaatatgga tctgcagata 7500

gattagatac agctgtggaa aagttaacaa ggtggacaaa tagcttatca acattcaagg 7560

atgtatgcac catgctaaag ttctcgatgg ttaatgattc taaagggatt aagacatata 7620

aagctaatgg agcagatttt cattcattat cagcaagcga gatcatgact tcacaaggat 7680

ttgatttgtt tgaacttcta aaactaataa aagcctgtga agcatgccac actagtaact 7740

cagttttgaa ccttattgca tttaaaaaca taaaaaataa aacatatata cctggattta 7800

agaagaattt agccaatact gttcacttca actatgaaat gaaattgaat aatgaagtta 7860

tgaacaaatt ttatgaatat aagacaattt ctttacaaga tataaaagtt tctgatagag 7920

tcagatcagt cctggaaaca catggattca gtgtaactgg ccaaaatttg aaactagaag 7980

aagtagattt agaattgaat cctgtagagg ttatagatga ttcttctact tatgaccaga 8040

tagctagaca gatgagattg acaaaaaaga aaagttcata tttaataccg gctaatactt 8100

tattgttagg agaattaatg aaatttctaa tgttatgtat caatggtgaa gagcatgaca 8160

taatgaaatt attaagaagc cattttgcaa tgagacctgt gagagaaaat aggttacttt 8220

gcattcaaga tgctgtcatg acttgcagga taagtggttt tatccaaagg cattttctaa 8280

atgacaagaa agagatagtc ttgttgggtg tctcagatag cttagaaaat tttatttctc 8340

tggcgacccc aaactattca gtccaggagc cattcaaagc agaagcctta ataaaaaaag 8400

ctctgctgga ggtatatgat tcagggaaat acaaaatatt gaaaaacata agaagctata 8460

tagtagcaga tgtagagttt cttacagaga aatgtttata ttctaacata actaatgagg 8520

aaataagtag catgatattt aatgctgtta gcaaaattga tttggagtta gctaatctga 8580

aaccaaaaaa gaggaaaaag agattagatt tacatgatgt atttaataaa tttttaggat 8640

caggtgctac ttcttcagaa caagaataaa agggaggtaa tttatgggcg agagaaaagg 8700

gataagacat agaatagata atttgaattc taaagcagga aaagagcatg acatcaaaag 8760

gtatgttttt ggaacaaaaa gataaaaaat attctaactc agtctgaaat gaaatgtagt 8820

tatagtttct cggcctgaca atagagtctg aaatagtatt tctttttaaa tctgagactg 8880

tgtttagaat actttaattt tgttgctcga ttgctct 8917

<210> 4

<211> 275

<212> PRT

<213> nucleocapsid protein (N)

<400> 4

Met Ser Asn Val Lys Gln Leu Thr Glu Lys Lys Ile Lys Asp Leu Leu

1 5 10 15

Ala Gly Gly Thr Ala Asp Val Glu Ile Glu Thr Glu Asp Ser Thr Pro

20 25 30

Gly Phe Ser Phe Lys Ala Phe Tyr Glu Asn Asn Lys Asn Ile Glu Ile

35 40 45

Thr Phe Met Asn Cys Leu Asn Ile Leu Lys Cys Arg Lys Gln Ile Phe

50 55 60

Ala Ala Cys Lys Asn Gly Lys Tyr Val Phe Cys Gly Lys Thr Ile Val

65 70 75 80

Ala Thr Ser Ala Asp Val Gly Pro Glu Asp Trp Thr Phe Lys Arg Thr

85 90 95

Glu Ala Phe Ile Arg Thr Lys Met Val Ser Met Val Glu Lys Asn Lys

100 105 110

Asn Glu Ala Ala Lys Gln Asp Met Tyr Ala Lys Ile Met Glu Leu Pro

115 120 125

Leu Val Ala Ala Tyr Gly Leu Asn Val Pro Ala Ser Phe Asp Ser Cys

130 135 140

Ala Leu Arg Met Met Leu Cys Ile Gly Gly Pro Leu Pro Leu Leu Ser

145 150 155 160

Ser Ile Lys Gly Leu Ala Pro Ile Ile Phe Pro Leu Ser Tyr Tyr Gln

165 170 175

Asn Val Lys Lys Glu Lys Leu Gly Ile Lys Asn Phe Ser Thr Tyr Glu

180 185 190

Gln Val Cys Lys Val Ala Lys Val Leu Ser Ala Ser Gln Ile Glu Phe

195 200 205

Lys Asp Asp Leu Asp Ser Leu Phe Lys Ala Ala Val Lys Met Leu Ser

210 215 220

Glu Ser Asn Pro Gly Thr Ala Ser Ser Asn Ser Leu Lys Lys Tyr Asp

225 230 235 240

Asp Gln Val Lys Tyr Met Asp Arg Val Phe Ser Ala Ser Leu Thr Val

245 250 255

Asp Asp Tyr Gly Glu His Ser Lys Lys Ser Ser Lys Pro Ser Ser Ser

260 265 270

Leu Glu Val

275

<210> 5

<211> 439

<212> PRT

<213> non-structural proteins (NSs)

<400> 5

Met Ser Thr Ala Lys Ser Ala Ala Ser Glu Phe Ile Lys Ser Tyr Gly

1 5 10 15

Thr Arg Asp Asn Arg Ala Ile Asn Asp Cys Tyr Ser Val Phe Ser Gly

20 25 30

Glu Gly Val Asn Phe Leu Asn Leu Phe Met His Asn Asn Ala Gly Ile

35 40 45

Lys Ser Ala Phe Ser Ile Asn Asp Leu Gly Arg Asn Glu Asp Val Lys

50 55 60

Ile His Glu Ala Glu Val Ile Asp Ser Cys His Asp Tyr His Tyr Phe

65 70 75 80

Glu Lys Phe Gly Leu Asp Ile Thr Phe Cys Glu His Glu Met Asn Leu

85 90 95

Ile Val Arg Lys Pro Gly Ile Lys Asn Thr Gly Cys Lys Phe Ser Met

100 105 110

His Ser Gln Ile Phe Asn Pro Asn Pro Asp Met Leu Ala Leu Ile Pro

115 120 125

Gly Thr Val Ser Glu Glu Asp Phe Tyr Glu Lys Ser Lys Ile Arg Ile

130 135 140

Asp Gly Leu Leu Pro Ser Gly Trp Cys Leu Asp Glu Cys Trp Lys Asn

145 150 155 160

Asn Phe Tyr Ile Ala Thr Asn Gly Asp Phe Asn Leu Asp Tyr Gly Phe

165 170 175

Ser Val Met Gly Lys Thr Thr Ser Tyr Trp Arg Glu Ser Ile Pro Lys

180 185 190

Glu Lys Ile Leu Ser Val Lys Gln Lys Cys Leu Pro Asp Asn Thr Val

195 200 205

Pro Thr Asn Arg Leu Leu Ser Thr Ser Thr Val Lys Gly Ile Gln Leu

210 215 220

Gly Ser Glu Leu Ala His Asp Thr Thr Val Ile Leu Ser Ser Lys Gln

225 230 235 240

Asn Leu Asn Thr Asp Leu Lys Ser Gln Tyr Arg Ile Ser Phe His Gly

245 250 255

Ile Gln Glu Glu Gly Ala Phe Ser Arg Thr Phe Cys Ile Pro Phe Glu

260 265 270

Asn Lys Ser Arg Met Ile Cys Leu Tyr Ala Lys Thr Val Ala Asp Asn

275 280 285

Ser Asn Glu Arg Thr Thr Leu Ile Ile Lys Val Val Thr Lys Thr Val

290 295 300

Asp Ser His Leu Val Ile Pro Ile Arg Asn His Ile Asn Cys Gly Arg

305 310 315 320

Lys Val Gly Ala Arg Ile Gly Leu Val Asp Phe Leu Glu Ser Asp Pro

325 330 335

Asn Tyr Asn Gln Met Ile Val Lys Glu Leu Leu Thr Val His Thr Gln

340 345 350

Phe Ala Ile Asn Leu Ser Glu Val Met Lys Arg Pro Ile Ile Val Phe

355 360 365

Lys Met Tyr Asp Arg Glu Leu Arg Asn Gly His Thr Glu Val Leu Gly

370 375 380

Arg Val Leu Asn Tyr Gln Ile Asp Asp Glu Gly Asn Ile Tyr Phe Leu

385 390 395 400

Ser Lys Thr Leu Glu Val Leu Pro Lys Ser Leu Ser Thr Leu Ser Tyr

405 410 415

Leu Gly Ser Ile Ala Pro Val Gln Trp Lys Glu Ser Leu Glu His Gln

420 425 430

His Phe Ala Val Lys Ala Glu

435

<210> 6

<211> 307

<212> PRT

<213> Athletic protein (NSm)

<400> 6

Met Ser Arg Phe Ser Asn Val Leu Glu Ser Leu Arg Pro Val Lys Ser

1 5 10 15

Ser Asn Lys Glu Leu Val Pro Ala Val Lys Thr Glu Asn Asn Arg Asn

20 25 30

Ile Leu Ala Arg Asn Val Ser Arg Lys Asp Ile Asp Asp Ala Ile Met

35 40 45

Asn Lys Ala Lys Thr Ile Asn Gly Lys Gln Tyr Val Ser Gly Gly Asp

50 55 60

Ser Ser Val Leu Gly Thr Tyr Ser Ser Glu Leu Ala Val Glu Ala Thr

65 70 75 80

Ser Asp Asp Ile Leu Ser Arg Leu Val Val Glu Gln Ser Thr His Leu

85 90 95

Ser Ser Trp Lys Asn Asp Ser Leu Val Gly Asn Gly Asn Glu Lys Val

100 105 110

Ser Phe Thr Ile Asn Ile Met Pro Thr Trp Asn Ser Gly Arg Arg Phe

115 120 125

Met His Ile Ser Arg Leu Ile Val Trp Ile Val Pro Thr Ile Pro Asp

130 135 140

Ala Lys Asn Asn Ile Lys Ala Thr Leu Val Asp Pro Asn Lys Met Thr

145 150 155 160

Arg Asp Glu Lys Ile Val Ile Ser Arg Gln Ser Ser Leu Lys Asp Pro

165 170 175

Met Cys Phe Ile Phe His Leu Asn Trp Ser Phe Pro Lys Glu Arg Asn

180 185 190

Thr Pro Lys Gln Cys Met Gln Leu Asn Leu Thr Ser Asp Glu Lys Tyr

195 200 205

Ala Lys Gly Val Ser Phe Ala Ser Val Met Tyr Ser Trp Val Lys Asn

210 215 220

Phe Cys Asp Thr Pro Ile Ala Ala Glu Ser Asn Thr Cys Asp Val Val

225 230 235 240

Pro Ile Asn Arg Ala Lys Val Ile Gln Ser Ala Ala Leu Ile Glu Ala

245 250 255

Cys Lys Leu Met Ile Pro Lys Gly Thr Gly Gly Lys Gln Ile Ala Asn

260 265 270

Gln Ile Lys Ser Leu Gln Lys Ala Ala Glu Lys Leu Ala Leu Glu Ala

275 280 285

Glu Asn Glu Asp Gly Asn Ala Asp Leu Glu Ile Glu Met Asp Asn Ser

290 295 300

Ile Asp Ile

305

<210> 7

<211> 1121

<212> PRT

<213> glycoprotein (Gn or Gc)

<400> 7

Met Lys Lys Tyr Tyr Leu Leu Val Tyr Cys Leu Gly Leu Leu Ser Leu

1 5 10 15

Phe Phe Val Ser Glu Val Tyr Leu Leu Asn Gln Ala Asp Asn Ser Val

20 25 30

Gln Leu Arg Lys Ile Gln Asp Arg Tyr Lys Val Asp Asp Pro Asp Asp

35 40 45

Leu Ile Glu Glu Asp Glu Val Tyr Ile Thr Glu Thr Ser Pro Pro Lys

50 55 60

Gln Lys Lys Leu Ser Arg Ile Leu Arg Asp Asp Glu Thr Thr Thr Gln

65 70 75 80

Ser Thr Ile Ser Glu Leu Ser Cys Asp Asn Phe Glu Lys Arg Tyr Cys

85 90 95

Met Ile Lys Gly Val Ser Asp Phe Asn Ala His Tyr Gln Ile Asp Ser

100 105 110

Gly Lys Glu Ile Val Ser Cys Ile Ser Ser Ser Ala Asn Ile Phe Asp

115 120 125

Ile Cys Gln Tyr Glu Lys Glu Phe Lys Lys Met Lys Phe Lys Asp Leu

130 135 140

Pro Val Val Pro Val Leu Lys Leu Glu Asn Lys Lys Val Leu Glu Ile

145 150 155 160

Gly Thr Lys Phe Phe Phe Val Asp Lys Ser Ser Asn Pro Ile Asn Ile

165 170 175

Asp Pro Arg Ile Asn Leu Lys Ser Pro Thr Val Ala Arg Leu Ser Val

180 185 190

Arg Leu Ser Gly Asp Cys Lys Ile Asn Gln Ile Ser Met Ser Ser Pro

195 200 205

Tyr Gln Ile Lys Leu Arg Ser Glu Glu Ser Val Gly Val Leu Ile Arg

210 215 220

Asn Ile Lys Asn Ser Lys Pro Glu Asn Val Lys Pro Ile Val Gly Asp

225 230 235 240

Ser Thr Ile Asn Phe Lys Pro Glu Glu Leu Asp Gly Asn His Phe Leu

245 250 255

Leu Cys Gly Asp Lys Ser Ser Leu Ile Ala Lys Val Asp Ile Pro Val

260 265 270

Arg Asn Cys Val Ser Lys Tyr Ser Gly Glu Pro Lys Lys Ile Phe Phe

275 280 285

Cys Ala Asn Phe Ser Tyr Phe Lys Trp Ile Phe Val Phe Leu Met Val

290 295 300

Ala Phe Pro Ile Ser Trp Leu Ile Trp Lys Thr Lys Asp Ala Leu Ser

305 310 315 320

Ile Trp Tyr Asp Ile Ile Gly Ile Leu Thr Tyr Pro Ile Leu Trp Thr

325 330 335

Ile Asn Trp Leu Trp Pro Tyr Phe Pro Leu Lys Cys Arg Val Cys Gly

340 345 350

Cys Phe Ser Phe Leu Thr His Ser Cys Ser Glu Glu Cys Val Cys Asn

355 360 365

Gln Asp Lys Ala Ser Lys Ser His Thr Ser Glu Cys Tyr Leu Phe Thr

370 375 380

Lys Asp Lys Ala Glu Trp Lys Lys Leu Thr Leu Ile Gln Gln Phe Gln

385 390 395 400

Phe Thr Ile Asn Thr Lys Leu Ser Thr Asn Phe Leu Val Phe Leu Thr

405 410 415

Lys Met Ile Leu Ala Ser Ile Leu Ile Ser Tyr Ile Pro Ser Ser Ile

420 425 430

Ala Leu Lys Gln Pro Asn Leu Cys Val Glu Lys Cys Tyr Tyr Asn Leu

435 440 445

Asn Leu Asp Ser Leu Thr Thr Asp Lys Phe Gly Ile Thr Asp Asn Gly

450 455 460

Tyr Glu Thr Cys Glu Cys Ser Ile Gly Asn Val Ile Thr Glu Thr Val

465 470 475 480

Tyr Arg Ser Gly Val Pro Met Ser Arg Ala Thr Ser Leu Asn Asp Cys

485 490 495

Ile Leu Gly Ser Asp Leu Cys Met Val Ser Asn Asn Gln Ala Gln Asn

500 505 510

Leu Phe Ala Cys Arg Asn Gly Cys Asn Ser Leu Arg Ser Ile Lys Asn

515 520 525

Ile Pro Asp Thr Lys Phe Asn Lys Tyr Tyr Lys Gly Gln Ser Phe Lys

530 535 540

Gly Asn Leu Thr Ser Leu Lys Ile Ala Asn Arg Leu Arg Glu Gly Tyr

545 550 555 560

Met Asp Ser Pro Thr Glu Ser Lys Ile Leu Glu Glu Glu Ser Ala Arg

565 570 575

Glu Tyr Lys Phe Tyr Lys Ser Leu Lys Val Asp Asp Ile Pro Pro Glu

580 585 590

Asn Leu Met Pro Arg Gln Ser Leu Val Phe Ser Thr Glu Val Asp Gly

595 600 605

Lys Tyr Arg Tyr Leu Leu Glu Met Asp Ile Lys Thr Asn Thr Gly Ser

610 615 620

Ile Tyr Leu Leu Asn Asp Asp Ala Ala His Ser Pro Met Glu Phe Met

625 630 635 640

Ile Tyr Ile Lys Ser Val Gly Val Glu Tyr Asp Ile Arg Tyr Lys Tyr

645 650 655

Ser Thr Ala Lys Ile Asp Thr Thr Val Ala Asp Tyr Leu Val Thr Cys

660 665 670

Thr Gly Ser Cys Thr Glu Cys Val Lys Gln Lys Pro Lys Val Gly Val

675 680 685

Leu Asp Phe Cys Val Thr Pro Thr Ser Trp Trp Gly Cys Glu Glu Leu

690 695 700

Gly Cys Leu Ala Ile Asn Glu Gly Ser Ile Cys Gly His Cys Thr Asn

705 710 715 720

Ile Tyr Asp Leu Ser Ser Leu Val Asn Val Tyr Gln Val Val Glu Ser

725 730 735

His Val Ala Ala Glu Val Cys Val Lys Ser Leu Asp Gly Tyr Ser Cys

740 745 750

Lys Lys His Ser Asp Arg Ser Pro Ile Gln Thr Asp Tyr Tyr Gln Leu

755 760 765

Asp Met Ser Ile Asp Leu His Asn Asp Tyr Met Ser Thr Asp Lys Leu

770 775 780

Phe Ala Val Thr Lys Gln Gln Lys Ile Leu Thr Gly Asn Ile Ala Asp

785 790 795 800

Leu Gly Asp Phe Ser Gly Ser Ser Phe Gly His Pro Gln Ile Thr Met

805 810 815

Asp Gly Ile Pro Leu Ser Val Pro Ala Thr Leu Ser Gln Asn Asp Phe

820 825 830

Thr Trp Ser Cys Ser Ala Val Gly Asp Lys Lys Val Asn Ile Lys Gln

835 840 845

Cys Gly Leu Tyr Thr Tyr Ser Asn Ile Tyr Val Leu Ser Pro Ser Lys

850 855 860

Asp Leu Ser Tyr Leu Asp Glu Asn Asn Asn Lys Leu Tyr Met Glu Lys

865 870 875 880

Asp Phe Leu Val Gly Lys Leu Lys Met Ile Ile Asp Met Pro Lys Glu

885 890 895

Met Phe Lys Lys Ile Pro Thr Lys Pro Ile Leu Ser Glu Val Arg Met

900 905 910

Val Cys Ser Gly Cys Ser Gln Cys Ala Ala Gly Ile Asn Cys Asn Ile

915 920 925

Thr Tyr Thr Ser Asp Thr Thr Phe Ser Ser Arg Leu Met Met Asp Thr

930 935 940

Cys Ser Phe Lys Ser Asp Gln Ile Gly Thr Phe Leu Gly Pro Asn Glu

945 950 955 960

Lys Ser Ile Lys Ala Tyr Cys Ser Gly Glu Ile Ala Asp Lys Ser Leu

965 970 975

Lys Leu Ile Pro Glu Asp Gln Glu Glu Leu Thr Val Asp Ile Gln Val

980 985 990

Asp Glu Phe Thr Gln Val Asp Gln Asp Thr Ile Ile His Phe Asp Asp

995 1000 1005

Lys Ser Ala His Asp Glu Asn Thr His Asn Ser Asp Thr Ser Ile

1010 1015 1020

Ser Ser Leu Trp Asp Trp Ile Lys Ala Pro Phe Asn Trp Ile Ala

1025 1030 1035

Ser Phe Phe Gly Asn Phe Phe Asp Leu Val Arg Ile Ile Leu Val

1040 1045 1050

Ile Ala Ala Ala Cys Val Gly Leu Tyr Ile Val Ser Ser Ile Phe

1055 1060 1065

Lys Leu Ser Arg Thr Tyr Tyr Val Asp Lys Arg Arg Gln Lys Leu

1070 1075 1080

Glu Asp Val Val Glu Ser Leu Glu Ser Ser Val Leu Leu Thr Ser

1085 1090 1095

Phe Ala Glu Ser Asp Gln Thr Arg Lys Arg Arg Ser Pro Pro Lys

1100 1105 1110

Gly Tyr Asp Phe Ser Leu Asp Ile

1115 1120

<210> 8

<211> 2878

<212> PRT

<213> replicase (RdRp)

<400> 8

Met Asn Ile Gln Thr Ile Asn Ser Phe Leu Asp Thr Ser Gly Glu Ile

1 5 10 15

Thr Val Tyr Leu Gln Thr Ile Lys Asp Glu Ile Met Ser Met Ser Asn

20 25 30

Ile Val Asp Arg Ser Tyr Pro Glu Gly Lys Gly Val Ile Glu Ser Gly

35 40 45

Leu Asn Ser Ile Met Ser Met Asn Tyr Ile Leu Asp Lys Ile Glu Asp

50 55 60

Met Lys Ser Arg Tyr His Gln Ser Ser Ser Ser Ile Asp Gln Lys Glu

65 70 75 80

Met Glu Thr Glu Ile Phe Leu Phe Leu Asp Lys Tyr Lys Glu Leu Glu

85 90 95

Leu Met Arg His Asp Leu Phe Gly Val Leu Ala Gly Arg Arg Leu His

100 105 110

Phe Ala Pro Lys His Arg Ser Asp Val Phe Leu Lys Asp Cys Leu Met

115 120 125

Ser Tyr Ile Glu Phe Cys Asn Ser Ser Thr Ser Ile Val Asn Pro Ile

130 135 140

Val Asp Ile Asp Asp Leu Lys Glu Lys Leu Val Phe Gln His Leu Thr

145 150 155 160

Pro Asp Asn Tyr Thr Ile Tyr Lys Glu Thr Lys Gly Glu Lys Ala Cys

165 170 175

Leu Met Ile Tyr Asp Trp Lys Val Ser Ile Asp Ala Met Ser Glu Thr

180 185 190

Lys Thr Ser Glu Asn Tyr Tyr Thr Ser Val Trp Lys Thr Phe Lys Asp

195 200 205

Ile Thr Ile Asp Glu Gln Pro Phe Leu Glu Arg His Pro Ile Phe Ile

210 215 220

Thr Ile Val Ile Leu Asn Pro Ser Gly Asn Met Pro Ile Val Ala Thr

225 230 235 240

Thr Ser Arg Val Ile Gln Glu Phe Arg Asn Ser Pro Tyr Ser Asn Phe

245 250 255

Asn Asp Arg Lys Lys Ala Ala Thr Ala Ala Lys Leu Ile Ser Val Asn

260 265 270

Ser Leu Gly Gln Leu Ile Gly Leu Gly Ala Asp Cys Phe Arg Arg Phe

275 280 285

Tyr Ser Glu Thr Gln Val Phe Lys Asn Ser Leu Leu Ala Lys Val Gly

290 295 300

Glu Tyr Met Asn Arg Ser Ser Glu Val Phe Phe Ser His Trp Ser Tyr

305 310 315 320

Glu Tyr Arg Lys Thr Asn Leu Ser Asn Asn Gln Ile Ser Gln Asp Ile

325 330 335

Val Asp Leu Ile Asp Ala Met Pro Asn Asp Ala Ile Lys Lys Asp Met

340 345 350

Leu Val His Phe Leu Phe Gly Ser Tyr Leu Tyr Phe Lys Glu Thr Met

355 360 365

Ser Asp Leu His Leu Lys Asp Arg Phe Glu Gly Tyr Lys Ala His Cys

370 375 380

Glu Met Met Gly Ile Glu Pro Ile Glu Thr Glu Glu Lys Leu Lys Glu

385 390 395 400

Tyr Leu Asp Lys Asn Glu His Leu Phe Glu Asn Leu Tyr Glu Gly His

405 410 415

Leu Gly Lys Ile Lys Ser Asp Val Leu Phe Arg Lys Ser Lys Glu Ile

420 425 430

Glu Ile Glu Ser Ile Glu Ser Ala Phe Asn Ile Asn Ala Glu Glu Tyr

435 440 445

Gln Arg Glu Tyr Pro Gly Cys Phe Thr Asn Asp Leu Gln Glu Thr Lys

450 455 460

Thr Asn Phe Ser Ile Cys Trp Ser Pro Met Thr Gln Glu Leu Glu Met

465 470 475 480

Asn Glu Arg Asn Tyr Asn Asn Ala Ile Ile Gln Ser Phe Arg Asp Thr

485 490 495

Phe Thr Asp Gln Val Lys Leu Ile His His Lys Ser Tyr Gly Gly Ser

500 505 510

Arg Met Asp Thr His Phe Ser Ile Thr Leu Phe Asn Leu Val Lys Ala

515 520 525

Cys Leu Val Asp Leu Ser Thr Asp Thr Thr Gly His Ser Lys Ala Ala

530 535 540

Leu Glu Asp Val Val Asp Ile Lys Asp Gly Ser Ile Gln Val Lys Arg

545 550 555 560

Thr Glu Thr Ser Gln Lys Trp Lys Asp Ile Gly Lys Val Lys Thr Arg

565 570 575

Asn Gly Asn Glu Phe Thr Ile Ser Ser Ala Ser Asp Asn Glu Ala Arg

580 585 590

Lys Asn Phe Phe Lys Gly Leu Ser Leu Met Tyr Val Asp Met Gly Lys

595 600 605

Lys Lys Lys Ile Asp Ile Lys Asn Asp Leu Lys Ser Lys Ile Gln Glu

610 615 620

Ser Met Ala Glu Lys Lys Asp Glu Glu Ile Ser Gly Pro Ser Gly Glu

625 630 635 640

Tyr Asp Val Ser Lys Ala Asp Ile Pro Phe Gln Ile Ser Ile Lys Gly

645 650 655

Ile Thr His Lys Lys Lys Leu Ile Arg His Asp Asn Pro Asp Val Lys

660 665 670

Tyr His Cys Glu Ser Met Ile Glu Ala Met Tyr Val Leu Tyr Gly Met

675 680 685

Asp Glu Arg Lys Asp Lys Asn Ser Lys Ile Asn Ser Val Tyr Asn Glu

690 695 700

Tyr Cys Ser Asn Pro Ser Ser Leu Phe Ala Lys Gly Asn Leu Ile Glu

705 710 715 720

Thr Glu Met Ile Ile Ala Lys Asn Ile His Glu Val Ser Lys Glu Leu

725 730 735

Ser Val Tyr Thr Tyr Ser Glu Asp Met Met Gln Met Ala Lys Gly Leu

740 745 750

Met Val Ala Asp Arg Phe Met Lys Lys Thr Asp Phe Lys Ile Leu Thr

755 760 765

Cys Ala Asn Thr Ser Met Leu Cys Leu Ala Phe Lys Gly Asp Gly Ile

770 775 780

Asn Thr Gly Lys Ser Gly Val Pro Tyr Ile Thr Leu His Arg Val Glu

785 790 795 800

Lys Asp Ile Gln Pro Tyr Phe Ala Ser Leu Tyr Thr Lys Glu Leu Ile

805 810 815

Val Ser Phe Lys Ser Asp Asn Tyr Phe Ile Asn Ile Met Arg Pro Gln

820 825 830

Arg Leu Asn Gln Val Arg Leu Leu Ser Leu Phe Lys Ala Pro Ser Lys

835 840 845

Val Pro Val Leu Phe Ser Gln Tyr Ser Leu Leu Ser Thr Glu Ile Lys

850 855 860

Lys Trp Leu Asn Gln Ser Ser Val Asp Val Phe Thr Cys Pro Glu Asn

865 870 875 880

Lys Ile Gln Tyr Leu Gln Lys Ile Leu Phe Ser Ser Val Ile Ile Gly

885 890 895

Thr Val Thr Lys Leu Ser Arg Met Gly Ile Phe Asp Phe Met Arg Tyr

900 905 910

Ala Gly Phe Leu Pro Leu Ser Asp Tyr Ser Asn Ile Lys Glu Tyr Ile

915 920 925

Ala Glu Lys Phe Asp Pro Asp Ile Thr Asn Val Ile Asp Cys Phe Phe

930 935 940

Val Ser Gly Ile Lys Asn Leu Leu Leu Lys Met Glu Gly Ile Asn Leu

945 950 955 960

Ser Asn Ser Ile Lys Pro Leu Thr Ile Asp Gln Glu Asn Asp Met Ser

965 970 975

Gly Gly Ile Ser Asp Leu Asp Ile Val Cys Pro Ile Thr Gly Ser Thr

980 985 990

Leu Lys Thr Ile Glu Cys Leu Tyr Asn Asn Val Tyr Leu Ala Ile Tyr

995 1000 1005

Met Met Pro Lys Ser Leu His Thr His Val His Asn Leu Thr Thr

1010 1015 1020

Leu Leu Ser Val Pro Ala Glu Trp Glu Ile Lys Phe Arg Glu Lys

1025 1030 1035

Met Gly Phe Leu Ile Gly Asp Glu Ile Lys Pro Lys Lys Glu Met

1040 1045 1050

Phe Asn Asp Ser Gly Pro Phe Ser Ile Asn Gly Val Leu Asn Val

1055 1060 1065

Lys Thr Leu Phe Asp Tyr Tyr Lys Lys Asn Ile Ile Asn Val Gly

1070 1075 1080

Ala Cys Arg Ser Asn Ile Glu Asp Lys Glu Asp Phe Leu Ser Ala

1085 1090 1095

Pro Tyr Lys Ile Lys Thr Leu Thr Ser Ser Lys Lys Cys Ser Lys

1100 1105 1110

Ala Asp Ile Ile Lys Asn Ser Glu Ile Val Ala Ala Leu Lys Ser

1115 1120 1125

Thr Phe Gly Lys Gln Pro Asp Asp Ile Lys Gly Ser Asp Leu Tyr

1130 1135 1140

Ile Leu Lys Gly Val Leu Lys Cys Phe Glu Glu Asp Arg Glu Ser

1145 1150 1155

Leu Ile Asn Phe Leu Glu Ile Glu Ser Leu Asp Glu Ser Lys Tyr

1160 1165 1170

Leu His Phe Phe Ser Asn Met Met Ser Gly Asn Asn Lys Val Leu

1175 1180 1185

Met Lys Thr Asn Gln Asp Lys Phe Tyr Tyr Lys Ser His Pro Leu

1190 1195 1200

Thr Val Glu Thr Phe Met Lys Val Arg Tyr Gly Tyr Phe Asn Thr

1205 1210 1215

Thr Thr Val Leu Thr Ser Lys Lys Val Ser Glu Glu Leu Tyr Asp

1220 1225 1230

Leu Ile Lys Glu Phe Asn Lys Ile Thr Glu Ile Asp Leu Glu Ala

1235 1240 1245

Leu Glu Lys Leu Gly Arg Gly Leu Lys Gly Asn Arg Val Thr Phe

1250 1255 1260

Ile Gln Leu Leu Glu Phe Ile Leu Met Lys Ser Arg Thr Asn Ala

1265 1270 1275

Gly Asn Thr Asp Phe Leu Val Ser Val Phe Glu Lys Met Gln Arg

1280 1285 1290

Thr Lys Met Asp Arg Glu Ile Tyr Leu Met Ser Met Lys Thr Lys

1295 1300 1305

Met Met Leu Tyr Phe Ile Glu His Thr Tyr Lys His Val Ala Gln

1310 1315 1320

Ser Asp Pro Ser Glu Ala Ile Ser Ile Ser Gly Asp Tyr Lys Ile

1325 1330 1335

Lys Asn Leu Ala Ser Leu Ser Tyr Asp Thr Ile Thr Asn Tyr Asn

1340 1345 1350

Thr Ala Leu Gln Lys Asn Leu Glu Cys Lys Met Ala Phe Leu Ser

1355 1360 1365

Ala Asp Gln Ser Lys Trp Ser Ala Ser Asp Leu Thr Tyr Lys Tyr

1370 1375 1380

Ile Leu Ala Val Ile Met Asn Pro Ile Leu Thr Thr Gly Glu Ile

1385 1390 1395

Asn Leu Met Cys Glu Cys Ile Met Met Tyr Val Lys Leu Lys Arg

1400 1405 1410

Val Cys Ile Pro Thr Asp Ile Phe Leu Asn Leu Lys Arg Gly Gln

1415 1420 1425

Thr Gln Tyr Gly Ser Tyr Gly Thr Ala Leu Ser Met Leu Thr Asp

1430 1435 1440

Asn Leu Glu Thr Asn Thr Phe Pro Val Ser Met Asn Trp Leu Gln

1445 1450 1455

Gly Asn Leu Asn Tyr Leu Ser Ser Val Tyr His Ser Cys Ala Met

1460 1465 1470

Met Gly Tyr Glu Lys Ala Met Lys Lys Met Lys Asp Tyr Asp Phe

1475 1480 1485

Thr Ile Arg Trp Met Val His Ser Asp Asp Asn Ala Thr Ser Met

1490 1495 1500

Val Val Arg Gly Asp Ile Lys Lys Leu Leu Ser Ser Phe Asn Cys

1505 1510 1515

Ser Ser Leu Ser Glu Leu Leu Phe Arg Ser Ile Gln Ser His Phe

1520 1525 1530

Lys Ser Tyr Cys Ile Thr Leu Asn Pro Lys Lys Ser Tyr Ala Ser

1535 1540 1545

Glu Ser Glu Val Glu Phe Ile Ser Glu Arg Ile Ile Asn Gly Ala

1550 1555 1560

Val Ile Pro Leu Tyr Cys Arg His Leu Ala Asn Cys Ser Thr Glu

1565 1570 1575

Ser Ser His Asn Ser Tyr Phe Asp Asp Leu Met Ser Leu Ser Ile

1580 1585 1590

His Ile Thr Met Leu Leu Arg Lys Gly Cys Pro Asn Glu Leu Ile

1595 1600 1605

Pro Phe Ala Tyr Ala Ala Ile Gln Thr Gln Ser Leu Ser Ile Tyr

1610 1615 1620

Ser Met Leu Pro Gly Glu Glu Asn Asp Ile Ser Ser Ile Ile Lys

1625 1630 1635

Gly Thr Asn Phe Pro Leu Ser Lys Arg Glu Ile Pro Ile Cys Ala

1640 1645 1650

Gly Gly Trp Met Tyr Ala Pro Ile Glu Leu Leu Ser Ile Leu Gly

1655 1660 1665

Pro Ser Ser Asn Asp Gln Leu Ile Tyr Tyr Lys Ile Ile Leu Asp

1670 1675 1680

Phe Phe Asp Leu Arg Asp Phe Asn Ser Leu Lys Lys Ser Val Asn

1685 1690 1695

Ser Leu Gly Tyr Ile Asp Leu Arg Ile Asn Glu Leu Phe Lys Arg

1700 1705 1710

Ile Lys Tyr Gln Lys Thr Thr Cys Ala Asp Arg Lys Met Ile Cys

1715 1720 1725

Met Val Asn Leu Phe Lys Thr Ser Leu Met Ser Glu Asp Cys Asp

1730 1735 1740

Asn Leu Asn Val Gly Met Lys Phe Gln Ser Met Ile Thr Gln Ile

1745 1750 1755

Ile Lys Leu Pro Ser Phe Val Ser Glu Gly Ser Leu Leu Lys Asn

1760 1765 1770

Ser Ser Phe Gln Asp Phe Cys Lys Pro Phe Pro Asn Leu Lys Lys

1775 1780 1785

Asn Thr Asp Ile Leu Asp Ala Leu Lys Pro Ser Asn Leu Asn Glu

1790 1795 1800

Lys Asp Leu Glu Glu Val Ser Glu Ser Lys Phe Leu Ser Gln Ile

1805 1810 1815

Gln Leu Glu Glu Leu Ser Arg His Met Ser Cys His Pro Glu Ser

1820 1825 1830

Phe Leu Ile Ala Pro Met Asn Asp Lys Asp Tyr Ile Leu Thr Asn

1835 1840 1845

Leu Tyr Thr Tyr Ser Ser Val Ser Lys Arg Asn Gln Met Ser Asn

1850 1855 1860

Gln Ser Thr Glu Lys Leu Ala Leu Asp Arg Ile Leu Arg Ser Lys

1865 1870 1875

Ala Lys Thr Phe Leu Asp Pro Asn Ser Lys Glu Met Val Ser Tyr

1880 1885 1890

Lys Asp Asn Met Ser Met Lys Met Thr Glu Ile Met Asn Pro Thr

1895 1900 1905

Gly Asn Asp Phe Lys Ile Ile Asn Thr Ile Ser Gly Leu Met Val

1910 1915 1920

Arg Asp Met Asn Phe Glu Met Val Val Ser Leu Met Glu Asn Thr

1925 1930 1935

Val Ala Asn Ala Ser Ile Pro Lys Ala Asn Tyr Asn Phe Arg Trp

1940 1945 1950

Phe Ile Thr Glu Lys Val Pro Ser Val Ile Glu Gly Ser Pro Gly

1955 1960 1965

Leu Ile Val Met Ser Ala Val Tyr Gly Met Asp Tyr Leu Ile Glu

1970 1975 1980

Leu Gly Leu Lys Lys Leu Pro Leu Thr Glu Met Ser Ile Ser Ile

1985 1990 1995

Leu His Asp Ile Phe Gly Asn Arg Lys Thr Phe Asp Asp Val Lys

2000 2005 2010

Glu Cys Ile Gln Lys Thr Asn Asn Gln Tyr Lys Thr Glu Glu Phe

2015 2020 2025

Gln Asn Thr Asn Asp Leu Lys Arg Tyr Val Leu Ser Ile Asn Tyr

2030 2035 2040

Met Ile Gln Ser Gln Asn Lys Leu Leu Ser Ile Asn Thr Cys Phe

2045 2050 2055

Ser Arg Lys Asn Phe Pro Phe Tyr Ser Lys Tyr Asn Leu Gly Arg

2060 2065 2070

Thr Phe Ile Thr Asn Thr Leu Ala Ile Trp Ser Thr Ile Tyr Ser

2075 2080 2085

Arg Leu Thr Asn Ile Asn Phe Tyr Thr Asn Leu Asn Phe Val Ile

2090 2095 2100

Asp Arg Ser Ser Arg Leu Ile Ile Ser Leu Gln Arg Asp Met Asn

2105 2110 2115

Leu Glu Lys Leu Ile Asp Cys Cys Ala Tyr Val Ser Asp Arg Leu

2120 2125 2130

Gln Ser Leu Phe Pro Asp Met Thr Ile Asp Ser Ile Lys Gln Ile

2135 2140 2145

Leu His Arg Leu Asn Phe Asn Gly Val Asp Leu Met Lys Lys Met

2150 2155 2160

Lys Ser Glu Ile Thr Glu Val Lys Arg Ala Ile Asn Asn Ile Lys

2165 2170 2175

Thr Ser Thr His Val Thr Leu Ser Phe Arg Pro Gln Met Ile Ala

2180 2185 2190

Met Ser Lys His Ala Ala Trp Leu Tyr Asn Phe Gly Tyr Ile Asn

2195 2200 2205

Glu Lys Glu Phe Lys Phe Val Ile Asp Gln Ile Arg Gln Ser Glu

2210 2215 2220

Val His Tyr Ile Lys Thr Asp Glu Gln Asp Val Arg Gly Tyr Tyr

2225 2230 2235

Val Ser Gly His Thr Tyr Lys Ile Gly Ile Lys Thr Gln Tyr Asn

2240 2245 2250

Tyr Gly Gln Leu Ala Met Ser Asn Gln Asp Ile Ser Ile His Leu

2255 2260 2265

His Ser Pro Tyr Glu Tyr Gln Arg Glu Asp Asp Gly Lys Ile Trp

2270 2275 2280

Glu Val His Val Arg Ser Ala Tyr Lys Leu Leu Gln Lys Leu Leu

2285 2290 2295

Ile Asp Lys Gln Ser Ala Ile Lys Thr Phe Leu Asn Ile Arg Thr

2300 2305 2310

Glu Leu Met Pro Asn Gln Phe Cys Ile His Glu Ser Ser Asn Lys

2315 2320 2325

Thr Leu Leu Ile Leu Ile Asn Asp Thr Thr Lys Pro Ile Ala Leu

2330 2335 2340

Glu Arg Ile Lys Phe Lys Gly Asp Ile Lys Tyr Ile Pro Ser Ser

2345 2350 2355

Asp Phe Ala Trp Asn Met Met Ser Asn Gln Ser Lys Tyr Lys Leu

2360 2365 2370

Arg Pro Ala Glu Thr Gly Glu Cys Tyr Thr Glu Leu Tyr Lys Met

2375 2380 2385

Ile Asp Ser Asn Asp Gly Leu Met Asn Thr Ile Leu Ser Asn Leu

2390 2395 2400

Lys Lys Ser Leu Glu Tyr Gly Asn Glu Met Glu Asn Leu Ile Glu

2405 2410 2415

Gln Ser Ile Lys Asp Ile Asp Asp Glu Glu Thr Ile Asp Tyr Ile

2420 2425 2430

Arg Asp Ser Val Asp Gln Ile His Lys Leu Ala Val Glu Gly Leu

2435 2440 2445

Glu Thr Ser Lys Thr Ser Glu Glu Phe Glu Asn Tyr Leu Lys Arg

2450 2455 2460

Val Asp Phe Gln Ser Thr Val Asp Phe His Lys Asp Leu Leu Glu

2465 2470 2475

Gln Ile Val Glu Glu Lys Tyr Gly Ser Ala Asp Arg Leu Asp Thr

2480 2485 2490

Ala Val Glu Lys Leu Thr Arg Trp Thr Asn Ser Leu Ser Thr Phe

2495 2500 2505

Lys Asp Val Cys Thr Met Leu Lys Phe Ser Met Val Asn Asp Ser

2510 2515 2520

Lys Gly Ile Lys Thr Tyr Lys Ala Asn Gly Ala Asp Phe His Ser

2525 2530 2535

Leu Ser Ala Ser Glu Ile Met Thr Ser Gln Gly Phe Asp Leu Phe

2540 2545 2550

Glu Leu Leu Lys Leu Ile Lys Ala Cys Glu Ala Cys His Thr Ser

2555 2560 2565

Asn Ser Val Leu Asn Leu Ile Ala Phe Lys Asn Ile Lys Asn Lys

2570 2575 2580

Thr Tyr Ile Pro Gly Phe Lys Lys Asn Leu Ala Asn Thr Val His

2585 2590 2595

Phe Asn Tyr Glu Met Lys Leu Asn Asn Glu Val Met Asn Lys Phe

2600 2605 2610

Tyr Glu Tyr Lys Thr Ile Ser Leu Gln Asp Ile Lys Val Ser Asp

2615 2620 2625

Arg Val Arg Ser Val Leu Glu Thr His Gly Phe Ser Val Thr Gly

2630 2635 2640

Gln Asn Leu Lys Leu Glu Glu Val Asp Leu Glu Leu Asn Pro Val

2645 2650 2655

Glu Val Ile Asp Asp Ser Ser Thr Tyr Asp Gln Ile Ala Arg Gln

2660 2665 2670

Met Arg Leu Thr Lys Lys Lys Ser Ser Tyr Leu Ile Pro Ala Asn

2675 2680 2685

Thr Leu Leu Leu Gly Glu Leu Met Lys Phe Leu Met Leu Cys Ile

2690 2695 2700

Asn Gly Glu Glu His Asp Ile Met Lys Leu Leu Arg Ser His Phe

2705 2710 2715

Ala Met Arg Pro Val Arg Glu Asn Arg Leu Leu Cys Ile Gln Asp

2720 2725 2730

Ala Val Met Thr Cys Arg Ile Ser Gly Phe Ile Gln Arg His Phe

2735 2740 2745

Leu Asn Asp Lys Lys Glu Ile Val Leu Leu Gly Val Ser Asp Ser

2750 2755 2760

Leu Glu Asn Phe Ile Ser Leu Ala Thr Pro Asn Tyr Ser Val Gln

2765 2770 2775

Glu Pro Phe Lys Ala Glu Ala Leu Ile Lys Lys Ala Leu Leu Glu

2780 2785 2790

Val Tyr Asp Ser Gly Lys Tyr Lys Ile Leu Lys Asn Ile Arg Ser

2795 2800 2805

Tyr Ile Val Ala Asp Val Glu Phe Leu Thr Glu Lys Cys Leu Tyr

2810 2815 2820

Ser Asn Ile Thr Asn Glu Glu Ile Ser Ser Met Ile Phe Asn Ala

2825 2830 2835

Val Ser Lys Ile Asp Leu Glu Leu Ala Asn Leu Lys Pro Lys Lys

2840 2845 2850

Arg Lys Lys Arg Leu Asp Leu His Asp Val Phe Asn Lys Phe Leu

2855 2860 2865

Gly Ser Gly Ala Thr Ser Ser Glu Gln Glu

2870 2875

Claims (3)

1. The tomato spotted wilt virus gene is characterized in that the tomato spotted wilt virus is a pepper yellow ringspot virus, the base sequence of the gene is shown as SEQ ID NO. 2, and the gene can encode the movement protein NSm and glycoprotein Gn or Gc of the virus; the amino acid sequence of the virus gene sequence code shown as SEQ ID NO. 2 is shown as SEQ ID NO. 6 and SEQ ID NO. 7.

2. A detection kit for detecting a tomato spotted wilt virus gene is characterized in that the tomato spotted wilt virus gene is a pepper yellow ringspot virus gene shown as SEQ ID NO. 2, and the kit contains 50 mu L of a sense primer with the concentration of 100pmol, 50 mu L of an antisense primer with the concentration of 100pmol and 2.5ml of a probe 50 mu L, RT-PCR reaction solution with the concentration of 100 pmol; the sequence of the sense primer is 1320-1340 in SEQ ID No. 2, the sequence of the antisense primer is the complementary sequence of 2700-2720 in SEQ ID No. 2, and the sequence of the probe is 1986-2200 in SEQ ID No. 2.

3. The detection kit according to claim 2, characterized in that the operating steps are as follows:

extracting total RNA of a disease sample, removing a disease plant, grinding by liquid nitrogen, extracting the total RNA of the disease sample, taking 12 muL of total RNA as a template, mixing 1 muL of 100 muM downstream primers, quickly inserting the DNA into ice for 5min after pre-denaturation at 70 ℃ for 10min, adding 4 muL of 5 xRT Buffer, 2 muL of 10mmol/L dNTP, 1 muL of 5U/mu L M-MLV, 1 muL of 40U/muL RNase Inhibitor, and extending for 90min at 42 ℃; using cDNA as template or PCR amplification; the reaction system is 50 muL: 3 muL of cDNA, 1 muL of upstream primer, 1 muL of downstream primer, dNTP1 muL, rTaq 1 muL, 10' buffer 5 muL, H₂O38 muL; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min for 30s, and cyclic amplification for 30 times; finally, extending for 10min at 72 ℃;

the PCR product is recovered by using a UNIQ-10 column PCR purification kit or a UNIQ-10 column gel recovery kit, the recovered fragment is used as a probe or is connected to a pMD18-T vector, and the connection system is as follows: 2.5. mu.L of Ligation mix, 0.5. mu.L of pMD18-T, 2. mu.L of PCR-recovered product, ligated overnight at <16 ℃; the DNA was transformed into E.coli DH 5. alpha. by heat shock at 42 ℃ and positive clones were screened by colony PCR and sequenced.