WO2020096301A1 - Recombination vector based on newcastle disease virus isolated from domestic wild ducks, and method for producing same - Google Patents

Abstract

The present invention pertains to: a recombination vector produced by inserting the full-length gene of a novel Newcastle disease virus strain KU001 (Accession Number: KCTC18794P) isolated from domestic wild ducks; and a method for producing same.

Description

Recombinant vector of Newcastle disease virus isolated from wild ducks in Korea and its preparation method

The present invention relates to a recombinant vector prepared by inserting a full-length gene of a novel Newcastle disease virus isolated from a domestic wild duck and a method for manufacturing the same.

Newcastle disease virus (NDV) is a negative sense single-stranded RNA virus that is a contagious virus belonging to the genus Paramyxoviridae, Mononegavirales, and Avulavirus to be. The virus is a highly contagious virus that occurs well in poultry, especially chickens and ducks. In Korea, it has a high incidence and is recommended to be vaccinated against NDV worldwide by governments. The length of the genome is 15,186 nucleotides. NDV contains six transcription units that encode other viral proteins in the genome: nucleocapsid protein (NP), phosphoprotein (P), matrix (M), fusion protein (F), and hemagglutinin neuraminidase ( HN) and polymerase (L). Synthesis of mRNA encoded by these transcription units catalyzes viral RNA-dependent RNA polymerase in the cytoplasm of infected cells. All transcription units are flanked by conserved gene initiation and gene end sequences. In the course of viral replication, mRNA synthesis causes a gradient in mRNA transcription, where genes close to the 3 'end, such as NP, are transcribed more than genes located far from the 3' end, such as L. Thus, the expression level of the new transcription unit will depend on its position relative to the 3 'end.

The Newcastle disease virus has an envelope, the HN protein that allows the virus to bind to the host cell and the F protein that causes the fusion of the envelope with the host cell. F protein and HN protein are glycoproteins that are distributed on the surface of the envelope.

F protein

Type membrane glycoprotein (type

membrane glycoprotein) to form a trimeric structure. The F protein is made into an inactive precursor form (F0) and is cleaved into the active forms F1 and F2 while traveling through the Golgi membranes. This process exposes the hydrophobic domain at the amino terminus of the F1 subunit, which plays an important role in the biological activity of the mature protein. The hydrophobic domain, called the fusion peptide, is highly conserved in the paramyxovirus F protein and is believed to be directly involved in mediating membrane fusion. The paramyxovirus F protein has several common structural forms, including seven repeats (heptad repeats) and two regions likely to form an alpha helix structure. Of the two repeats, the longest seven repeat A (heptad repeat A) is adjacent to the hydrophobic fusion peptide at the amino terminus of F1, and the seven repeats B are in close contact with the upper part of the transmembrane region. The 7 repeats B consisted of a sequence of highly conserved leucine or isoleucine every 7 residues.

NDV infects various poultry species. It causes severe respiratory and nervous system diseases in many types of poultry species. The strains of NDV were classified according to their virulence: a lentogenic strain causing an unsigned infection in birds; Mesogenic strains causing respiratory diseases in birds; A velogenic strain that causes a blistering infection, often accompanied by signs of the nervous system in birds. Several pharmacopathogenic strains of NDV have recently been used as live attenuated vaccines in chickens.

Ideal vaccines to protect the general public or poultry from infectious diseases are vaccines that are easily prepared and rapidly trigger a strong protective immune response. Live virus vaccines have been demonstrated to be effective against a variety of viral diseases and have induced strong persistent humoral and cellular immune responses. Recombinant NDV is a good candidate for use as a live virus vaccine vector because it is not pathogenic to humans, humans have no previous immunity to it, and easily grows in embryonated eggs for large-scale production.

Republic of Korea Registered Patent 10-1099629

An object of the present invention is to provide a recombinant vector in which the full-length gene of Newcastle disease virus is inserted, which is more productive than the B1 strain or Lasota strain used as a conventional Newcastle disease virus vaccine strain, and a method for manufacturing the same.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, a method for preparing a recombinant vector in which the full-length gene sequence of the Newcastle disease virus KU001 strain (Accession No: KCTC18794P) is inserted, comprising the following steps, using cDNA using RNA extracted from Newcastle disease virus. Synthesizing; The nucleotide sequence encoding the NP protein, the base sequence encoding the P protein, the base sequence encoding the M protein, the base sequence encoding the F protein, the base sequence encoding the HN protein, and the base sequence encoding the L protein are sequentially Inserting into the plasmid; It includes, wherein the recombinant vector is made of a nucleotide sequence of SEQ ID NO: 1, a method for producing a recombinant vector is provided.

According to one side, the nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, the base encoding the M protein The sequence consists of the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, The nucleotide sequence encoding the L protein may be composed of the nucleotide sequence of SEQ ID NO: 7.

According to one side, the Newcastle disease virus KU001 strain may be isolated from domestic wild ducks.

According to another embodiment of the present invention, a full-length gene sequence of the Newcastle disease virus KU001 strain (Accession number: KCTC18794P) is inserted, and a recombinant vector consisting of the nucleotide sequence of SEQ ID NO: 1 is provided.

According to one side, the recombinant vector may be a plasmid vector.

The vector produced by the method for preparing a recombinant vector of the present invention includes a full-length gene of Newcastle disease virus, and thus is used as a backbone vector for the production of multivalent vaccines that can prevent diseases such as avian influenza as well as Newcastle disease. Can be used.

In addition, the recombinant virus prepared from the recombinant vector of the present invention has superior productivity compared to the B1 strain or the Lasota strain used as the existing Newcastle disease virus vaccine strain, thereby reducing production cost during mass production of the recombinant virus using the recombinant vector of the present invention. This is possible.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the invention.

1 is a schematic diagram of a recombinant vector of the present invention.

Figure 2 is a schematic diagram of the recombinant vector construction process of the present invention.

Figure 3 shows a phylogenetic tree analysis table using the fusion protein (F) sequence of Newcastle disease virus.

Figure 4a is a diagram showing a method for confirming whether or not the recombinant vector of the present invention is constructed using PCR technique, and Figures 4b to 4d are results of confirming fragments using P1 / P2, P4 / P6, and P10 / P12 primers, respectively. .

5A and 5B confirm whether a recombinant virus is produced from the recombinant vector of the present invention using RT-PCR technique.

6A and 6B confirm whether a recombinant virus is produced from the recombinant vector of the present invention using a Genetic tag (NotI).

Figure 7 shows the existing Newcastle disease virus (rNDV / B1 / wt (rB1 wt), rNDV / Lasota / wt (rLasota wt))

And the growth characteristics of the virus (rKU001) produced with the recombinant vector of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. The same reference numerals in each drawing denote the same members.

Various changes can be made to the embodiments described below. The examples described below are not intended to be limiting with respect to the embodiments, and should be understood to include all modifications, equivalents, or substitutes thereof.

The terms used in the examples are only used to describe specific examples, and are not intended to limit the examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described in the specification, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, actions, components, parts or combinations thereof are not excluded in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

In the present invention, the term "cDNA" refers to DNA complementary to mRNA, and preferably refers to DNA having a base sequence complementary to mRNA of Newcastle disease virus.

In the present invention, the term "vector" is a means for introducing a foreign gene and allowing the expression of the foreign gene, a plasmid vector capable of replicating itself, a cosmid vector, a bacteriophage vector, and an adenovirus. Vectors, retroviral vectors, adeno-associated virus vectors, and the like.

In the present invention, the term "restriction enzyme (restriction enzyme)" is an endonuclease (endonuclease) that is one of the nucleases that recognize a specific nucleotide sequence of DNA or RNA and cleaves the base chain. It means a special enzyme used to make.

In the present invention, the term "primer (primer)" refers to a DNA or RNA having a short gene sequence complementary to a specific gene sequence to be synthesized, means that it is synthesized for use in PCR, DNA sequencing, and the like.

In the present invention, the term "promotor (promotor)" refers to the region on the DNA that initiates transcription by specifically binding RNA polymerase. In the present invention, the promoter is not only a basic factor (basal element) required for transcription, but also expression of Enhancers that can be used for palpation and control may be further included.

According to an embodiment of the present invention, a method for preparing a recombinant vector in which the full-length gene sequence of the Newcastle disease virus KU001 strain (Accession No: KCTC18794P) is inserted, comprising the following steps, using cDNA using RNA extracted from Newcastle disease virus. Synthesizing; The nucleotide sequence encoding the NP protein, the base sequence encoding the P protein, the base sequence encoding the M protein, the base sequence encoding the F protein, the base sequence encoding the HN protein, and the base sequence encoding the L protein are sequentially Inserting into the plasmid; It includes, wherein the recombinant vector is made of a nucleotide sequence of SEQ ID NO: 1, a method for producing a recombinant vector is provided.

The nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence encoding the M protein is SEQ ID NO: 4 Consisting of the nucleotide sequence of the, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, encoding the L protein The base sequence may be composed of the base sequence of SEQ ID NO: 7.

The Newcastle disease virus KU001 strain is isolated from domestic wild ducks, and refers to a strain that has been granted accession number KCTC18794P from the Korea Research Institute of Bioscience and Biotechnology (181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea) as of October 31, 2019. The genotype corresponds to Genotype I (Fig. 3). Since the recombinant vector of the present invention contains the full-length gene of NDV / KU001 strain isolated from domestic wild ducks, it is possible to prepare a recombinant virus vaccine effective for the prevention of the Newcastle disease virus that is prevalent in Korea when it is used as a backbone vector.

According to another embodiment of the present invention, a full-length gene of the Newcastle disease virus KU001 strain (Accession number: KCTC18794P) is inserted, and a recombinant vector consisting of the nucleotide sequence of SEQ ID NO: 1 is provided. Here, the recombinant vector may be a plasmid vector, but a pSL1180 vector may be used, but is not limited thereto.

The recombinant vector of the present invention can be used to prepare a recombinant virus by reverse genetics. The method for producing a specific recombinant virus is as follows. First, in order to provide a T7 RNA polymerase, a virus (MVA / T7 virus) expressing T7 RNA polymerase in a Modified Vaccinia Ankara strain is infected with Hep-2 or A549 cells. Three plasmids (NP, P. L) expressing NDV RNA polymerase protein and the recombinant vector pNDV / KU001 of the present invention are simultaneously transfected into the Hep-2 or A549 cells. 24 hours after transfection, co-culture with chicken embryo fibroblast (CEF) cells is performed. After incubation for 48 hours or more, the cell supernatant is injected into a 10-day-old fertilized egg, and after 72 hours of incubation, urine fluid is obtained.

Hereinafter, experimental materials and methods used in the Examples of the present invention will be described in detail. However, the following examples are only illustrative of the contents of the present invention, and the scope of the invention is not limited by the examples.

Example 1: Construction of recombinant vector

To construct the recombinant vector of the present invention, RNA was extracted using TRIzol (Invitrogen, USA) from Newcastle disease virus KU001, and then reverse transcription PCR was performed with Superscript ^TM II Reverse Transcriptase (Invitrogen, USA) to synthesize cDNA. . Subsequently, 7 gene fragments (Section 1, T7 promoter to nucleotide 1,755 nucleotide sequence; fragment 2, nucleotide 1 nucleotide sequence to 3,321 sequence; fragment 3, nucleotide 1,755 nucleotide sequence to 6,580 sequence; fragment 4 , Nucleotides from 6,151 nucleotide sequence to 10,210 sequence; fragment 5, nucleotide 7,381 nucleotide sequence to 11,351 sequence; fragment 6, nucleotide 11,351 nucleotide sequence to 14,995 sequence; fragment 7, nucleotide 14,701 nucleotide sequence to 15,186 After obtaining the second sequence) by PCR method, hepatitis delta virus ribozyme, T7 terminator gene sequence was subsequently inserted into the segment 7 gene, and the entire gene sequence was sequentially inserted into the pSL1180 (Amersham Parmacia Biotech, UK) plasmid to pNDV / KU001 The vector was completed. Here, p of pNDV means plasmid.

Example 2: Validation of the construction of the recombinant vector

In order to verify the pNDV / KU001 vector constructed in Example 1, a PCR technique was performed as described in FIG. 4A. NP-P-M-F and HN-L sections were used as controls. The primers used were P1 / P2 (Fig. 4b), P4 / P6 (Fig. 4c), and P10 / P12 (Fig. 4d). Primer P1 used (forward direction; 5'-ACCAAACAGAGAATCCGTGAG-3 ') is the sequencing information in front of the NP gene of NDV, P2 (reverse direction; 5'-AGATGAGGGGGTTCGTTTGTAG-3') is the sequencing information prior to 1701 of NDV, P4 (forward direction; 5'-CGCGCCGTCGCAGTGACC-3 ') is 4292th, P6 (reverse direction; 5'-CGTTTCCCACCGAACCGC-3') is 7514th, P10 (forward direction; 5'- ACCGACCCCAACACAGTTCC-3 ') is 13825th , P12 (reverse direction; 5'- ACCAAACAAAGATTTGGTG-3 ') was designed based on the last sequence information of NDV, respectively. As a result, as shown in B to D of FIG. 4, it was confirmed that a pNDV / KU001 plasmid having an F protein coding base sequence and an HN protein coding base sequence linked thereto was constructed.

Example 3: Verification of the production of recombinant virus

It was confirmed whether the recombinant virus was actually produced using the recombinant vector constructed in Example 1 above. Hereinafter, r of rNDV represents recombinant, and rNDV means recombinant virus.

3-1. Verification of the production of recombinant rNDV / KU001 virus using RT-PCR technique

The rNDV / KU001 virus was prepared by the reverse genetics method described below. First, in order to provide a T7 RNA polymerase, a virus (MVA / T7 virus) expressing T7 RNA polymerase in a Modified Vaccinia Ankara strain was infected with Hep-2 or A549 cells. The pNDV / KU001 vector constructed in Example 1 and three plasmids expressing NP, P, and L proteins were transfected into infected Hep-2 or A549 cells. After incubation for more than 48 hours. Chicken embryo fibroblast (CEF) cells were added and co-cultured for 2-3 days. Next, the cell supernatant was obtained and inoculated into the allantoic cavity of 9-10-day-old chicken eggs, and again after 2-3 days, the allantoic fluid was obtained to perform the hemagglutination assay and hemagglutination-inhibition assay to confirm the presence of the virus.

In order to verify that the rNDV / KU001 virus was actually produced from the recombinant vector of the present invention, total RNA was extracted from the virus and then cDNA was prepared using random hexamer. Using the produced cDNA as a template, PCR was performed to amplify the HN gene (FIG. 5A), and comparative analysis was performed with the control rNDV / Lasota / wt using the restriction enzyme XbaI present only in the KU001 strain (FIG. 5B).

As a result, as shown in Figure 5, only the HN gene of the KU001 strain was cut, and it was confirmed that the virus produced therefrom was derived from the recombinant vector of the present invention.

3-2. Verification of the production of recombinant rNDV / KU001 virus using Genetic tag (NotI)

The rNDV / KU001 virus produced by the reverse genetics method has a NotI restriction enzyme recognition site that is not found in the wild type NDV KU001 strain, so NotI was used as a genetic marker to distinguish the wild type virus from the recombinant virus produced by the reverse genetics method. .

After extracting the RNA of the wild type virus and rNDV / KU001 virus, RT-PCR was performed to obtain a PCR gene product containing NotI restriction enzyme (FIG. 6A), and the gene product was treated with restriction enzyme NotI (FIG. 6B).

As a result, as shown in FIG. 6, it was confirmed that the PCR product was cleaved when the restriction enzyme NotI was treated in the case of the rNDV / KU001 virus. Through this, it was confirmed that the rNDV / KU001 virus produced by the reverse genetics method is not a wild type NDV KU001 strain, but is a new virus derived from the recombinant vector of the present invention.

Example 4: Evaluation of productivity of recombinant virus

The recombinant NDV KU001 virus prepared in Example 3 and the Newcastle disease virus B1 strain and Lasota strain used as existing vaccine strains were prepared, and each virus was infected with 10-day-old fertilized eggs at a titer of 100 TCID ₅₀ . After 12, 24, 48 and 72 hours from viral infection, urine was obtained from the fertilized egg, and the viral titer was measured by Hemagglutinin assay (HA), and the results are shown in FIG. 7.

As a result, it was confirmed that the growth in comparison with the titers of the recombinant virus of the present invention Lasota weeks compared to 10 ² times, about 10 weeks B1 ⁵ times higher as shown in Fig. These results suggest that the production cost can be reduced when the vaccine is mass-produced using the recombinant vector of the present invention.

As described above, although the embodiments have been described by a limited embodiment and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order than the described method, and / or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

<110> KOREA UNIVERSITY RESEARCH & BUSINESS FOUNDATION

<120> Newcastle disease virus backbone vector using NDV KU001 strain

         and method for preparing thereof

<130> FPC-2019-0157

<150> KR 10-2018-0134494

<151> 2018-11-05

<150> KR 10-2019-0138666

<151> 2019-11-01

<160> 13

<170> KoPatentIn 3.0

<210> 1

<211> 15186

<212> DNA

<213> Newcastle disease virus

<400> 1

accaaacaga gaatccgtaa ggtacgatag aaggcgaagg agcaatcgaa gtcgtacggg 60

tagaaggtgt gaatctcgag tgcgagcccg aagctcaaac tcgagagagc cttctgccaa 120

aatgtcttct gtattcgatg agtacgagca gctcctcgcg gctcagactc gccccaatgg 180

agctcatggc ggaggagaga aggggagcac cttaaaggta gaagtcccgg tattcactct 240

caacagtgat gacccagaag atagatggaa ctttgcagtg ttttgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cacttaggca aggtgctctc atatctctct tatgttccca 360

ctctcaagtg atgaggaacc atgttgccct tgcggggaaa cagaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttaccaacgg cgtgccccag ttcaacaaca ggagtggagt 480

gtctgaagag agagcacaga gatttatgat gatagcaggg tctctccctc gggcatgcag 540

caacggtacc ccgttcgtca cagctggggt tgaagatgat gcaccagaag acattactga 600

taccctggag aggatcctct ctatccaggc tcaagtatgg gtcacggtgg caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga atcaataagt acatgcagca 720

aggcagggtc cagaagaagt acatcctcca ccccgtatgc aggagcgcaa tccaactcac 780

aatcagacag tctctggcgg tccgcatctt tttggttagc gagcttaaga gaggccgcaa 840

cacggcaggt gggacctcca cctattacaa cttggtgggg gatgtagact catacatcag 900

gaacactggg ctaactgcat tcttcctgac acttaaatat ggaattaaca ccaagacatc 960

agcccttgca cttagcagcc tctcaggcga tatccagaaa atgaagcagc tcatgcgctt 1020

gtatcggatg aaaggagata atgcgccgta catgacattg ctcggtgaca gtgaccagat 1080

gagctttgca cctgccgagt atgcacaact ttactccttt gccatgggta tggcatcagt 1140

cctagataaa ggaactagca aataccaatt tgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtacg ctcaggctca aggaagtagc atcaatgagg atatggccgc 1260

cgagctaaag ctaaccccag cagcaaggag aggcctggca gctgctgccc aaagagtgtc 1320

tgaggagacc agcagcatgg acatgcccac ccaacaagcc ggggtcctca ctggactcag 1380

cgacggaggc tcccaagccc cccaaggtgc actgaacaga tcacaagggc aaccggacac 1440

cggggatggg gagacccaat ttctggatct gatgagagcg gtggcaaata gcatgagaga 1500

agcgccaaac tctgcgcagg gcacccctca accggggcct cccccaaccc ctgggccctc 1560

tcaagacaat gacaccgact gggggtactg accgacagca cccagtttgc ttctatgagg 1620

tcatcccaat tcctctgccc acaccccacc cctcaatccg caatcccgca tggtcaaacc 1680

cacaaacgaa cccccctgtc tccctcctct cccccagccc cacaacccca cctgcccagg 1740

gcaacatagg tacaatgcga cccactaata atcaatacag ggccaaagaa attagaaaaa 1800

agtacgggta gaagggagac attcagagat cagggcgagt cacccgggtc tctgctctcc 1860

cttctaccta gtggattagg atggagatgg ccacctttac agatgcggag atcgacgagc 1920

tatttgagac cagtggaact gtcattgaca gcataattac ggcccaggga aaaccagtag 1980

agactgttgg aaggagtgca atcccacaag gcaaaactaa ggctttgagc gcagcatggg 2040

agaagcatgg gagcatccag tcaccagcca gccaagacac ccctgatcga caggacagat 2100

cagataaaca actgtccaca cccgagcaag cgagtccaaa cgacagcccc ccagccacat 2160

ccactgacca gcctcccact caggctgcag atgaggccgg cgatacacag ctcaagaccg 2220

gagcaagcaa ctctctgctg tcgatgcttg ataaactcag caataagtca tctaatgcta 2280

aaaagggccc agggtcgagc cctcaagaaa ggcatcatca acgtctgact caacaacagg 2340

ggagtcaaca aagccgcgga aacagccaag agagaccgca gaaccaggcc aaggccatcc 2400

ctggaaacca ggtcacagac gcgaacacag catatcatgg acaatgggag gagtcacaac 2460

tatcagctgg tgcaacccat catgctctcc gatcagagca gagccaagac aatactcctg 2520

cacctgtgga tcatgtccag ctacctgtcg actttgtgca ggcgatgatg tctatgatgg 2580

aggcgatatc acagagggta agtaaagttg actatcagct ggaccttgtc ttgaaacaga 2640

catcttctat ccccatgatg cggtctgaaa tccagcagct gaaaacgtct gttgcggtca 2700

tggaagccaa tttgggcatg atgaagatcc tggaccctgg ttgtgccaac gtttcatctc 2760

taagtgatct acgggcagtt gcccgatccc acccggtttt aatttctggc cccggagacc 2820

catctcctta tgtgacccaa gggggcgaaa tggcactcaa taaactttcg caaccggtgc 2880

aacacccctc tgaattgatt aaacccgcca cggcaagcgg gcctgatata ggagtggaga 2940

aagacactgt ccgtgcattg atcatgtcac gccctatgca tccgagctct tcagctaggc 3000

tcttgagcaa actggacgca gccggatcga ttgaggaaat cagaaaaatc aagcgccttg 3060

cactgaatgg ctaatcacca ccgcaacccg cagcagatcc ctgtccaccc agcaccacac 3120

ggtatctgca ccaagctcct ctctgcaaac ccaaggtcca acaccccgag cgacaaccct 3180

gtcctgcttc ctctgcccca ctaaatgatc gcgcagctgc aatcaattca gctatattaa 3240

ggattaagaa aaaatacggg tagaatcgga gtgccccgat tgtgccaaga tggactcatc 3300

taggacaatc gggctgtact ttgattctac ccttccttct agcaacctgc tagcattccc 3360

gatagtccta caagacacag gggacgggaa gaagcaaatc gccccgcaat acaggatcca 3420

gcgtcttgac tcgtggacag acagcaaaga agactcggta ttcatcacca cctatggatt 3480

catctttcag gttgggaatg aagaagccac tgtcggcatg atcaatgata atcccaagcg 3540

cgagttactt tccactgcca tgctatgcct agggagtgta ccaaatgtcg gagatcttgt 3600

tgagctggca agggcctgcc tcactatggt ggtaacatgc aagaagagtg caactaacac 3660

cgagagaatg gtcttctcag tagtgcaggc accccaggtg ctgcaaagct gtagggttgt 3720

ggcaaacaaa tactcgtcgg tgaatgcagt caagcacgtg aaagcaccag agaagattcc 3780

tgggagcgga accctagagt acaaagtgaa ctttgtctct ctgaccgtgg tgccaagaaa 3840

ggacgtctac aagataccaa ctgcagcact taaggtctct ggctcaagtc tgtacaatct 3900

tgcgctcaat gtcactattg atgtggaggt agacccgaag agcccgttgg tcaaatccct 3960

ttccaagtcc gacagtgggt actatgctaa tctcttctta catattgggc ttatgtccac 4020

tgtagataag aaggggaaga aagtgacatt tgacaagctg gaaaggaaga taaggagact 4080

tgatctatct gtagggctta gtgacgtgct cggaccttcc gtgcttgtaa aggcgagagg 4140

tgcacggact aagctgctgg cacctttctt ctctagcagt gggacagcct gctatcccat 4200

agcaaatgcc tctcctcagg tggccaagat actctggagc caaaccgcgt acctgcggag 4260

tgtaaaagtc attatccaag cgggcaccca gcgtgctgtc gcagtgaccg ccgaccacga 4320

ggttacctct actaagctgg agaaggggca taccattgcc aaatacaatc ccttcaagaa 4380

ataggctgca tctctgagat tgcactccgc ccatcttccc ggatcaccat gacactaaat 4440

aatgatctgt cttgattact tatagttagt tcgcctgtct atcaaattag aaaaaacacg 4500

ggtagaagat tctggatccc ggttggcgcc ttcaaggtgc aagatgggct ccagatcttc 4560

taccaggatc ccagtacctc tcatgctgac cgtccgagtc atgttggcac tgagttgcgt 4620

ctgtccgacc agcgcccttg atggcaggcc tcttgcagct gcagggattg tggtaacggg 4680

agacaaagca gtcaacatat acacctcatc tcagacaggg tcaatcataa tcaagttact 4740

cccaaatatg cccaaggata aagaggcgtg tgcaaaagcc ccgttggagg catacaacag 4800

gacattgact actttgctca ccccccttgg tgattctatc cgtaggatac aagagtctgt 4860

gaccacgtcc ggaggaggga aacagggacg tcttataggc gccattatcg gtggtgtagc 4920

tctcggggtt gcaaccgctg cacagataac agcagcctcg gctctgatac aagccaatca 4980

aaatgctgcc aacatactcc ggctaaaaga gagcattgct gcaaccaatg aggctgtgca 5040

cgaggtcact aatggattat cacaactagc agtggcagtt gggaagatgc agcaatttgt 5100

taatgaccag tttaataaaa cagctcagga attggactgt ataaaaatta cacagcaggt 5160

tggtgtagaa ctcaacctgt acctaactga attgactaca gtattcgggc cacaaatcac 5220

ttcccctgcc ttaactcagc tgactatcca ggcgctttac aatctagctg gtgggaatat 5280

ggattacttg ttgactaagt taggtgtggg gaacaaccaa ctcagctcat taattagtag 5340

tggcctgatc accggcaacc ctattctgta cgactcacag actcaactct tgggtataca 5400

ggtaacccta ccctcagtcg ggaacctaaa taatatgcgt gccacctacc tggaaacctt 5460

gtctgtaagt acaaccaaag gatttgcctc agcacttgtc ccaaaagtag tgacacaggt 5520

cggttccgtg atagaagagc ttgacacctc gtactgtata gagaccgatt tggatctata 5580

ttgtacaaga atagtgacat tccctatgtc tcctggtatt tattcctgtt tgagtggcaa 5640

tacatctgct tgcatgtact caaagactga aggcgcactc actacgccgt atatgaccct 5700

caaaggctca gttattgcta actgtaagat gacaacatgt agatgtgcag accccccggg 5760

tatcatatcg caaaattatg gagaagctgt gtctctaata gataggcaat catgcaatat 5820

cttatcctta gacgggataa ctttgaggct cagtggggaa tttgatgcaa cttatcaaaa 5880

gaatatctca atacaagatt ctcaagtaat agtgacaggc aatcttgata tctcgactga 5940

gcttgggaat gtcaacaact cgataagtaa tgctttggat aagttagagg aaagcaacag 6000

caaactagat aaggtcaatg tcaaactgac cagcacatcc gctcttatta cctatatcgt 6060

tttaactgtc atatctcttg tatgtggtat acttagcctg gttctagcat gctacctgat 6120

gtacaagcaa aaggcgcaac agaagacctt gttgtggctt gggaataata ccctagacca 6180

gatgagggcc actacaaaaa tgtgaatgcg gatgagaggc agaaacatcc ccaatagcag 6240

tttgtgtgta aagtctgaca gcctgttaat tagaagaatt aagaaaaaac taccggatgt 6300

agatgaccaa agggcgatat acgggtagaa cggtcgggga ggccgtccct caatcgggag 6360

ccgggcctca caacatccgt tctaccgcat caccaatagc agttttcagt catggaccgc 6420

gcagttagcc aagttgcgct ggagaatgat gaaagagagg caaagaatac atggcgcttg 6480

gtattccgga tcgcaatcct actctcaacg gtggtgacct tagccatctc tgcagccgcc 6540

cttgcatata gcatggaggc cagcacacct agcgatcttg taggcatacc gactgcgatc 6600

tctagagcag aggaaaagat tacatctgca ctcggttcca atcaagatgt agtagatagg 6660

atatataagc aggtggccct cgaatctcca ctggcattgc taaacaccga atctacaatt 6720

atgaacgcaa taacgtctct ctcttatcga atcaatgggg ccgcaaatag cagcggatgt 6780

ggagcaccca ttcatgatcc agattatatt ggaggaatag gtaaagaact tattgtagat 6840

gatgctagcg acgtcacatc atactatccc tctgcgttcc aagaacacct gaactttatc 6900

ccggcgccta ctacaggatc aggttgcact cggataccct catttgacat gagcgctacc 6960

cactactgtt atactcacaa tgtgatatta tctggctgca gagatcactc gcactcacat 7020

caatatttag cacttggtgt gcttcggaca tctgcaacag ggagggtatt cttttccact 7080

ctgcgttcca tcaatctgga tgacacccaa aatcggaagt cttgcagtgt gagtgcaacc 7140

cccttgggtt gtgatatgct gtgctctaaa gtcacagaga ctgaagaaga ggattataac 7200

tcagctatcc ccacgtcgat ggtacatgga aggttagggt tcgacggcca ataccacgag 7260

aaggacctag atgtcacaac actattcgag gactgggtgg caaactaccc aggagtaggg 7320

ggcgggtctt ttattgacaa ccgcgtatgg ttcccagttt acggagggct aaaacccaat 7380

tcgcccagtg acaccgcaca agaagggaaa tatgtaatat acaagcgata caatgacaca 7440

tgtccagatg agcaagatta tcagattcaa atggctaagt cttcatataa gcctgggcgg 7500

tttggaggga aacgcgtaca gcaggccatc ttatctatca aagtgtcaac atccttgggc 7560

gaggacccgg tactgactgt accgcccaac acagtaacac tcatgggggc cgaaggcaga 7620

gttctcacag tagggacatc tcatttcctt tatcagcgag ggtcatcata cttctcccct 7680

gccctactat atcctatgat agtcagcaac aaaacagcca ctcttcatag tccttataca 7740

ttcaatgcct tcactcgacc aggtagtgtc ccttgccagg cttcagcaag atgccctaac 7800

tcatgtgtta ccggagtcta tactgatcca tatcccttgg tcttctatag gaaccacacc 7860

ttgcgagggg tattcgggac gatgcttgat gataaacaag caagactcaa ccctgtatct 7920

gcagtatttg acagcatatc ccgcagtcgc ataacccggg tgagttcaag cagcaccaag 7980

gcagcataca caacatcaac atgttttaaa gttgtaaaga ccaataaaac ctattgtctc 8040

agcattgccg aaatatccaa taccctcttc ggggaattca gaatcgtccc tttactagtt 8100

gagattctca aggatgatgg ggttagagaa gccaggtcta gccggttgag tcaactgcga 8160

gagggttgga aagatgacat tgtatcacct atcttttgcg acgccaagaa tcaaactgaa 8220

taccggcgcg agctcgagtc ctacgctgcc agttggccat aatcagctag tgctaatgtg 8280

attagattaa gtcttgtcgg tagtcacttg attaagaaaa aatgtgggtg gtagcgggat 8340

ataaggcaaa acaactcaag gaggatagca cgggtaggac atggcgagct ccggtcccga 8400

gagggcggag catcagatta tcctaccaga gtcacacctg tcttcaccat tagtcaagca 8460

caaactactc tattactgga aattaactgg gctaccactc cctgacgagt gtgacttcga 8520

ccacctcatt ctcagccgac aatggaagaa aatacttgaa tcggcctccc ctgacactga 8580

gagaatgata aaacttggaa gggcagtgca ccagactctc aaccacaatt ccaagataac 8640

cggagtactc catcccaggt gtttagaaga attggctagt attgaggttc ctgactcaac 8700

caacaagttt cggaagatcg agaagaaaat ccaaattcac aacacaaggt atggagaact 8760

gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg ggatcatctt ggtctaataa 8820

tgtcccccgg tcagaagagt tcaacagcat ccgtacagat ccggcattct ggtttcactc 8880

aaaatggtcc acaactaagt ttgcatggct ccatataaaa cagattcaaa ggcatctgat 8940

tgtggcagca agaacaaggt ccgcagccaa caaattggtg acgctgaccc ataaggtagg 9000

ccaagtcttt gttactcctg agcttgtcat tgtgacacat acagatgaga acaagttcac 9060

gtgtcttacc caggaacttg tgttgatgta tgcagatatg atggagggca gagatatggt 9120

caacataata tcatccacgg cggcacatct caggagccta tcagagaaaa ttgatgacat 9180

tctgcggtta gtagatgccc tggcaaaaga tctgggtaat caagtctacg atgttgtagc 9240

actcatggag ggatttgcat acggcgccgt ccagctgctt gagccgtcag gtacattcgc 9300

aggggatttc ttcgcattca acctgcagga gctcaaagac actttgatcg gcctccttcc 9360

taaggatata gcagaatctg tgactcacgc aatagccact gtattctctg gcttagaaca 9420

aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg ggccacccat tacttgagtc 9480

ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca ccaaaaatgg tagactttga 9540

tatgatcctc caggtattgt ctttctttaa aggaacaatc atcaacggat acagaaagaa 9600

gaatgcaggt gtttggccac gtgtcaaagt agaaacgata tacgggaagg tcattgggca 9660

gctacacgct gattcagcgg agatttcaca cgatatcatg ttgagagagt acaagagttt 9720

atctgcgctt gaattcgagc catgtataga atacgaccct atcaccaatc tgagcatgtt 9780

tctaaaagac aaggcgatcg cacacccgaa agacaactgg ctcgccgcgt ttaggcgaaa 9840

ccttctctct gaggaccaga agaaacatgt aaaggaggca acctctacta accgtctctt 9900

gatagagttc ttagagtcaa atgattttga tccatataag gagatggaat atctgacgac 9960

ccttgagtac ctaagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt 10020

gaaggttaat gggcggattt ttgctaagct aacaaagaaa ttaaggaact gtcaagtgat 10080

ggcggaaggg atcttagctg accagattgc acctttcttt caagggaatg gggtcattca 10140

ggatagcata tctttaacca agagtatgct agcgatgagt caattgtctt tcaacagcaa 10200

taagaaacgt atcactgact gcaaagaaag agtagcctca aaccgcaatc acgatcaaaa 10260

gagcaagaat cgtcggagag ttgccacttt tataacgact gacctgcaaa agtactgtct 10320

taattggaga tatcagacaa tcaaactgtt cgctcatgcc atcaatcagc tgatgggctt 10380

acctcacttc ttcgaatgga ttcatctaag actaatggat actacgatgt ttgtaggaga 10440

ccctttcaat cccccaagtg acccaactga ctgtgatctc tcaagagtcc caaatgatga 10500

catatatatt gtcagtgcta gagggggtat tgagggatta tgtcagaagc tatggacaat 10560

gatctcaatt gctgcaatcc aacttgctgc agcaagatca cattgtcgcg tcgcctgtat 10620

ggtacagggt gacaatcaag taatagctgt aacgagagag gtaaggtcag atgactcccc 10680

ggaaatggtg ttaacacaat tgcatcaagc cagtgataat ttcttcaagg aattgattca 10740

tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa acaatcagat cagacacatt 10800

cttcatatac agcaaacgaa tattcaaaga tggagcaata ctcagtcaag tcctcaaaaa 10860

ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa aacaccgtaa tgtcctgtgc 10920

caacattgca tctactatag cacggctgtg cgagaacggg cttccaaagg atttctgtta 10980

ttacttaaac tacctgatga gttgcgtgca gacatacttt gattctgagt tttccatcac 11040

taacagctcg caccccgatt ctaaccagtc gtggattgaa gacatctctt ttgtgcactc 11100

atatgtcctg acccctgccc agctaggggg actgagcaac ctccaatact caaggctcta 11160

cacgaggaac atcggtgacc cgggaactac tgcttttgca gagatcaagc gattagaagc 11220

agtggggtta ctaagttcta gtattatgac taacatctta actaggccgc ctggaaatgg 11280

agattgggcc agtctgtgta acgaccctta ctctttcaat tttgagactg tcgcgagtcc 11340

aaatattgtc cttaagaaac atacacaaag agtcctattt gaaacttgtt caaatccctt 11400

attatctggc gtgcatacag aggataatga ggcagaagag aaggcgttgg ctgaattttt 11460

actcaatcaa gaagtaattc atccacgtgt cgcacatgct atcatggaag caagctctat 11520

aggtaggagg aagcagattc aagggcttgt tgacacaaca aacaccgtaa tcaagattgc 11580

attgactagg aggccacttg gcatcaagag gctgatgcgg atagttaact actcgagcat 11640

gcatgcaatg ctgtttagag acgatgtttt ctcatctaac aggtctaacc accccttagt 11700

ttcctctaat atgtgttctc tgacgctagc agactatgca cggaatagaa gctggtcacc 11760

attgacgggg ggtagaaaga tactgggtgt atctaatcct gatactatag aacttgtaga 11820

gggtgagatc cttagcgtca gcggaggatg cacaagatgt gacagcggag atgaacaatt 11880

cacttggttc catcttccga gcaatataga actgaccgat gacaccagca agaatcctcc 11940

gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg agggccgcct cgcttgcgaa 12000

aatagctcat atgtcaccac atgtgaaagc tgctctaagg gcatcatccg tgttgatctg 12060

ggcttatgga gacaacgaag taaattggac tgctgctctt aaaattgcaa gatctcggtg 12120

caatataaac tcagagtatc ttcgactatt gtccccctta cccacagctg ggaatctcca 12180

acatagactg gatgacggca taactcagat gacattcacc cctgcatctc tctacagggt 12240

gtcaccttat attcacatat ccaatgattc tcaaaggtta ttcacggaag aaggagtcaa 12300

agagggaaat gtagtttatc agcaaatcat gctcttgggt ttatctctaa tcgaatcact 12360

cttcccgatg acgacaacca ggacatacga tgagatcaca ttgcacctcc acagtaaatt 12420

tagctgctgt atcagggaag caccggttgc agttcctttc gagttactcg ggatggcacc 12480

agaactaagg acagtgacct caaataagtt tatgtatgat cctagtcctg tatcggaggg 12540

tgactttgcg agacttgact tagctatctt taagagttat gagcttaatc tagaatcata 12600

tcccacaata gagctaatga acattctttc aatatccagc gggaagttaa tcggccagtc 12660

tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa 12720

cacccggaat tggatcagcg aagctcagaa ttcagatgtg gtccgcctat tcgagtatgc 12780

agcacttgaa gtgcttctcg actgttctta tcagctctac tatctgagag taagaggcct 12840

agacaatatc gtgttgtata tgagtgactt atataagaat atgccaggaa ttctactttc 12900

caacattgca gctacaatat ctcatcccat cattcattca agattgcatg cagtaggcct 12960

ggtcaatcac gacgggtcac accaacttgc agacacagat ttcatcgaaa tgtctgcaaa 13020

actattagtc tcttgcactc gacgcgtggt ctcaggttta tatgcaggga ataagtatga 13080

tctgctgttc ccgtctgtct tagatgataa cctgagtgag aagatgcttc agctgatatc 13140

tcggttatgc tgcctgtata cggtgctctt tgctacaaca agagagatcc cgaaaataag 13200

aggcttatct gcagaagaga agtgttcagt acttactgag tacctactgt cagatgctgt 13260

gaaaccatta cttagttctg agcaagtgag ctctatcatg tctcctaaca tagttacgtt 13320

cccagctaat ctatattaca tgtctcggaa gagccttaat ttgattaggg aaagagagga 13380

cagggacact atcttggcat tgttgttccc ccaagagcca ctacttgagt tccccttagt 13440

acaagatatt ggcgctcgag tgaaagatcc attcacccga caacctgcgg cgtttttaca 13500

agaattagat ttgagcgctc cagcaaggta tgacgcattt acacttagtc aggttcattc 13560

tgaacacaca tcaccaaatc cggaggacga ctacttagta cgatacctgt tcagaggaat 13620

agggaccgcg tcctcctctt ggtataaggc atctcacctt ctttctgtac ctgaggtcag 13680

atgtgcaagg cacgggaatt ccttatactt ggcagaagga agcggagcca ttatgagtct 13740

tctcgaactg catgtgccgc atgagactat ctattacaat acgctcttct caaacgagat 13800

gaacccccca cagcggcatt tcggaccgac cccaacacag tttctgaatt cagttgttta 13860

taggaatcta caggcggagg taccatgtaa ggatggattt gtccaggagt tccgtccatt 13920

atggagagag aatacagaag aaagcgatct gacctcagat aaagcagtgg gttacatcac 13980

atctgcagtg ccctaccggt ctgtatcatt gctgcactgt gacattgaga ttcctccagg 14040

atccaatcaa agcttactgg atcaactggc taccaatcta tctctgattg ccatgcattc 14100

tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat gcaatgggat attacttcca 14160

tctactcatg aacttgttca ctccgtgttc tacgaaagga tatattctct ctaatggcta 14220

tgcatgtaga ggggatatgg agtgttacct ggtatttgtc atgggctatc gaggtgggcc 14280

tacatttgta catgaggtag tgaggatggc aaaaactcta gtgcagcggc acggtacact 14340

tttgtccaaa tcagatgaga tcacactgac taggttattt acctcacagc ggcagcgtgt 14400

aacagacatc ctatccagtc ctttaccgag actaataaag ttcttgagaa agaatatcga 14460

tactgcgcta attgaagccg ggggacaacc cgtccgtcca ttctgtgcag agagcttggt 14520

gaggacacta gcggacacaa ctcagatgac ccagatcatc gctagtcaca ttgacacagt 14580

cattcgatct gtgatctaca tggaggctga gggtgatctc gccgacacag tgttcttatt 14640

taccccctac aatctctcta cagacggtaa aaagagaaca tcacttaaac agtgcacaag 14700

gcagatctta gaggtcacaa tattgggtct tagagttgaa aatctcaata aagtaggtga 14760

tgtagtcagt ctagtactta aaggtatgat ttctctggag gacctgatcc ctctaagaac 14820

atacttgaag cgtagtacct gccctaagta tttgaagtct gttctaggta ttactaaact 14880

caaagaaatg tttacagaca cctctttatt atacttgact cgtgctcaac aaaaattcta 14940

catgaaaact ataggcaacg cagtcaaggg atactacagt aactgtgact cttaaaaata 15000

atcacatatt aataggctcc ttttctagtt aactgagccc ttgttgattt aatgatacta 15060

tattagaaaa aagttgcact ccgatccttt aggactcgtg ttcgaattca aataattgtc 15120

ttagaaaaaa gttgcgcgta attgttcttg aatgtagtcc tgtcattcac caaatctttg 15180

tttggt 15186

<210> 2

<211> 1470

<212> DNA

<213> Newcastle disease virus

<400> 2

atgtcttctg tattcgatga gtacgagcag ctcctcgcgg ctcagactcg ccccaatgga 60

gctcatggcg gaggagagaa ggggagcacc ttaaaggtag aagtcccggt attcactctc 120

aacagtgatg acccagaaga tagatggaac tttgcagtgt tttgtcttcg gattgctgtt 180

agcgaggatg ccaacaaacc acttaggcaa ggtgctctca tatctctctt atgttcccac 240

tctcaagtga tgaggaacca tgttgccctt gcggggaaac agaatgaggc cacactggct 300

gttcttgaga tcgatggttt taccaacggc gtgccccagt tcaacaacag gagtggagtg 360

tctgaagaga gagcacagag atttatgatg atagcagggt ctctccctcg ggcatgcagc 420

aacggtaccc cgttcgtcac agctggggtt gaagatgatg caccagaaga cattactgat 480

accctggaga ggatcctctc tatccaggct caagtatggg tcacggtggc aaaggccatg 540

actgcatatg agacagcaga tgagtcagaa acaagaagaa tcaataagta catgcagcaa 600

ggcagggtcc agaagaagta catcctccac cccgtatgca ggagcgcaat ccaactcaca 660

atcagacagt ctctggcggt ccgcatcttt ttggttagcg agcttaagag aggccgcaac 720

acggcaggtg ggacctccac ctattacaac ttggtggggg atgtagactc atacatcagg 780

aacactgggc taactgcatt cttcctgaca cttaaatatg gaattaacac caagacatca 840

gcccttgcac ttagcagcct ctcaggcgat atccagaaaa tgaagcagct catgcgcttg 900

tatcggatga aaggagataa tgcgccgtac atgacattgc tcggtgacag tgaccagatg 960

agctttgcac ctgccgagta tgcacaactt tactcctttg ccatgggtat ggcatcagtc 1020

ctagataaag gaactagcaa ataccaattt gccagggact ttatgagcac atcattctgg 1080

agacttggag tagagtacgc tcaggctcaa ggaagtagca tcaatgagga tatggccgcc 1140

gagctaaagc taaccccagc agcaaggaga ggcctggcag ctgctgccca aagagtgtct 1200

gaggagacca gcagcatgga catgcccacc caacaagccg gggtcctcac tggactcagc 1260

gacggaggct cccaagcccc ccaaggtgca ctgaacagat cacaagggca accggacacc 1320

ggggatgggg agacccaatt tctggatctg atgagagcgg tggcaaatag catgagagaa 1380

gcgccaaact ctgcgcaggg cacccctcaa ccggggcctc ccccaacccc tgggccctct 1440

caagacaatg acaccgactg ggggtactga 1470

<210> 3

<211> 1194

<212> DNA

<213> Newcastle disease virus

<400> 3

atggagatgg ccacctttac agatgcggag atcgacgagc tatttgagac cagtggaact 60

gtcattgaca gcataattac ggcccaggga aaaccagtag agactgttgg aaggagtgca 120

atcccacaag gcaaaactaa ggctttgagc gcagcatggg agaagcatgg gagcatccag 180

tcaccagcca gccaagacac ccctgatcga caggacagat cagataaaca actgtccaca 240

cccgagcaag cgagtccaaa cgacagcccc ccagccacat ccactgacca gcctcccact 300

caggctgcag atgaggccgg cgatacacag ctcaagaccg gagcaagcaa ctctctgctg 360

tcgatgcttg ataaactcag caataagtca tctaatgcta aaaagggccc agggtcgagc 420

cctcaagaaa ggcatcatca acgtctgact caacaacagg ggagtcaaca aagccgcgga 480

aacagccaag agagaccgca gaaccaggcc aaggccatcc ctggaaacca ggtcacagac 540

gcgaacacag catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccat 600

catgctctcc gatcagagca gagccaagac aatactcctg cacctgtgga tcatgtccag 660

ctacctgtcg actttgtgca ggcgatgatg tctatgatgg aggcgatatc acagagggta 720

agtaaagttg actatcagct ggaccttgtc ttgaaacaga catcttctat ccccatgatg 780

cggtctgaaa tccagcagct gaaaacgtct gttgcggtca tggaagccaa tttgggcatg 840

atgaagatcc tggaccctgg ttgtgccaac gtttcatctc taagtgatct acgggcagtt 900

gcccgatccc acccggtttt aatttctggc cccggagacc catctcctta tgtgacccaa 960

gggggcgaaa tggcactcaa taaactttcg caaccggtgc aacacccctc tgaattgatt 1020

aaacccgcca cggcaagcgg gcctgatata ggagtggaga aagacactgt ccgtgcattg 1080

atcatgtcac gccctatgca tccgagctct tcagctaggc tcttgagcaa actggacgca 1140

gccggatcga ttgaggaaat cagaaaaatc aagcgccttg cactgaatgg ctaa 1194

<210> 4

<211> 1095

<212> DNA

<213> Newcastle disease virus

<400> 4

atggactcat ctaggacaat cgggctgtac tttgattcta cccttccttc tagcaacctg 60

ctagcattcc cgatagtcct acaagacaca ggggacggga agaagcaaat cgccccgcaa 120

tacaggatcc agcgtcttga ctcgtggaca gacagcaaag aagactcggt attcatcacc 180

acctatggat tcatctttca ggttgggaat gaagaagcca ctgtcggcat gatcaatgat 240

aatcccaagc gcgagttact ttccactgcc atgctatgcc tagggagtgt accaaatgtc 300

ggagatcttg ttgagctggc aagggcctgc ctcactatgg tggtaacatg caagaagagt 360

gcaactaaca ccgagagaat ggtcttctca gtagtgcagg caccccaggt gctgcaaagc 420

tgtagggttg tggcaaacaa atactcgtcg gtgaatgcag tcaagcacgt gaaagcacca 480

gagaagattc ctgggagcgg aaccctagag tacaaagtga actttgtctc tctgaccgtg 540

gtgccaagaa aggacgtcta caagatacca actgcagcac ttaaggtctc tggctcaagt 600

ctgtacaatc ttgcgctcaa tgtcactatt gatgtggagg tagacccgaa gagcccgttg 660

gtcaaatccc tttccaagtc cgacagtggg tactatgcta atctcttctt acatattggg 720

cttatgtcca ctgtagataa gaaggggaag aaagtgacat ttgacaagct ggaaaggaag 780

ataaggagac ttgatctatc tgtagggctt agtgacgtgc tcggaccttc cgtgcttgta 840

aaggcgagag gtgcacggac taagctgctg gcacctttct tctctagcag tgggacagcc 900

tgctatccca tagcaaatgc ctctcctcag gtggccaaga tactctggag ccaaaccgcg 960

tacctgcgga gtgtaaaagt cattatccaa gcgggcaccc agcgtgctgt cgcagtgacc 1020

gccgaccacg aggttacctc tactaagctg gagaaggggc ataccattgc caaatacaat 1080

cccttcaaga aatag 1095

<210> 5

<211> 1662

<212> DNA

<213> Newcastle disease virus

<400> 5

atgggctcca gatcttctac caggatccca gtacctctca tgctgaccgt ccgagtcatg 60

ttggcactga gttgcgtctg tccgaccagc gcccttgatg gcaggcctct tgcagctgca 120

gggattgtgg taacgggaga caaagcagtc aacatataca cctcatctca gacagggtca 180

atcataatca agttactccc aaatatgccc aaggataaag aggcgtgtgc aaaagccccg 240

ttggaggcat acaacaggac attgactact ttgctcaccc cccttggtga ttctatccgt 300

aggatacaag agtctgtgac cacgtccgga ggagggaaac agggacgtct tataggcgcc 360

attatcggtg gtgtagctct cggggttgca accgctgcac agataacagc agcctcggct 420

ctgatacaag ccaatcaaaa tgctgccaac atactccggc taaaagagag cattgctgca 480

accaatgagg ctgtgcacga ggtcactaat ggattatcac aactagcagt ggcagttggg 540

aagatgcagc aatttgttaa tgaccagttt aataaaacag ctcaggaatt ggactgtata 600

aaaattacac agcaggttgg tgtagaactc aacctgtacc taactgaatt gactacagta 660

ttcgggccac aaatcacttc ccctgcctta actcagctga ctatccaggc gctttacaat 720

ctagctggtg ggaatatgga ttacttgttg actaagttag gtgtggggaa caaccaactc 780

agctcattaa ttagtagtgg cctgatcacc ggcaacccta ttctgtacga ctcacagact 840

caactcttgg gtatacaggt aaccctaccc tcagtcggga acctaaataa tatgcgtgcc 900

acctacctgg aaaccttgtc tgtaagtaca accaaaggat ttgcctcagc acttgtccca 960

aaagtagtga cacaggtcgg ttccgtgata gaagagcttg acacctcgta ctgtatagag 1020

accgatttgg atctatattg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080

tcctgtttga gtggcaatac atctgcttgc atgtactcaa agactgaagg cgcactcact 1140

acgccgtata tgaccctcaa aggctcagtt attgctaact gtaagatgac aacatgtaga 1200

tgtgcagacc ccccgggtat catatcgcaa aattatggag aagctgtgtc tctaatagat 1260

aggcaatcat gcaatatctt atccttagac gggataactt tgaggctcag tggggaattt 1320

gatgcaactt atcaaaagaa tatctcaata caagattctc aagtaatagt gacaggcaat 1380

cttgatatct cgactgagct tgggaatgtc aacaactcga taagtaatgc tttggataag 1440

ttagaggaaa gcaacagcaa actagataag gtcaatgtca aactgaccag cacatccgct 1500

cttattacct atatcgtttt aactgtcata tctcttgtat gtggtatact tagcctggtt 1560

ctagcatgct acctgatgta caagcaaaag gcgcaacaga agaccttgtt gtggcttggg 1620

aataataccc tagaccagat gagggccact acaaaaatgt ga 1662

<210> 6

<211> 1851

<212> DNA

<213> Newcastle disease virus

<400> 6

atggaccgcg cagttagcca agttgcgctg gagaatgatg aaagagaggc aaagaataca 60

tggcgcttgg tattccggat cgcaatccta ctctcaacgg tggtgacctt agccatctct 120

gcagccgccc ttgcatatag catggaggcc agcacaccta gcgatcttgt aggcataccg 180

actgcgatct ctagagcaga ggaaaagatt acatctgcac tcggttccaa tcaagatgta 240

gtagatagga tatataagca ggtggccctc gaatctccac tggcattgct aaacaccgaa 300

tctacaatta tgaacgcaat aacgtctctc tcttatcgaa tcaatggggc cgcaaatagc 360

agcggatgtg gagcacccat tcatgatcca gattatattg gaggaatagg taaagaactt 420

attgtagatg atgctagcga cgtcacatca tactatccct ctgcgttcca agaacacctg 480

aactttatcc cggcgcctac tacaggatca ggttgcactc ggataccctc atttgacatg 540

agcgctaccc actactgtta tactcacaat gtgatattat ctggctgcag agatcactcg 600

cactcacatc aatatttagc acttggtgtg cttcggacat ctgcaacagg gagggtattc 660

ttttccactc tgcgttccat caatctggat gacacccaaa atcggaagtc ttgcagtgtg 720

agtgcaaccc ccttgggttg tgatatgctg tgctctaaag tcacagagac tgaagaagag 780

gattataact cagctatccc cacgtcgatg gtacatggaa ggttagggtt cgacggccaa 840

taccacgaga aggacctaga tgtcacaaca ctattcgagg actgggtggc aaactaccca 900

ggagtagggg gcgggtcttt tattgacaac cgcgtatggt tcccagttta cggagggcta 960

aaacccaatt cgcccagtga caccgcacaa gaagggaaat atgtaatata caagcgatac 1020

aatgacacat gtccagatga gcaagattat cagattcaaa tggctaagtc ttcatataag 1080

cctgggcggt ttggagggaa acgcgtacag caggccatct tatctatcaa agtgtcaaca 1140

tccttgggcg aggacccggt actgactgta ccgcccaaca cagtaacact catgggggcc 1200

gaaggcagag ttctcacagt agggacatct catttccttt atcagcgagg gtcatcatac 1260

ttctcccctg ccctactata tcctatgata gtcagcaaca aaacagccac tcttcatagt 1320

ccttatacat tcaatgcctt cactcgacca ggtagtgtcc cttgccaggc ttcagcaaga 1380

tgccctaact catgtgttac cggagtctat actgatccat atcccttggt cttctatagg 1440

aaccacacct tgcgaggggt attcgggacg atgcttgatg ataaacaagc aagactcaac 1500

cctgtatctg cagtatttga cagcatatcc cgcagtcgca taacccgggt gagttcaagc 1560

agcaccaagg cagcatacac aacatcaaca tgttttaaag ttgtaaagac caataaaacc 1620

tattgtctca gcattgccga aatatccaat accctcttcg gggaattcag aatcgtccct 1680

ttactagttg agattctcaa ggatgatggg gttagagaag ccaggtctag ccggttgagt 1740

caactgcgag agggttggaa agatgacatt gtatcaccta tcttttgcga cgccaagaat 1800

caaactgaat accggcgcga gctcgagtcc tacgctgcca gttggccata a 1851

<210> 7

<211> 6615

<212> DNA

<213> Newcastle disease virus

<400> 7

atggcgagct ccggtcccga gagggcggag catcagatta tcctaccaga gtcacacctg 60

tcttcaccat tagtcaagca caaactactc tattactgga aattaactgg gctaccactc 120

cctgacgagt gtgacttcga ccacctcatt ctcagccgac aatggaagaa aatacttgaa 180

tcggcctccc ctgacactga gagaatgata aaacttggaa gggcagtgca ccagactctc 240

aaccacaatt ccaagataac cggagtactc catcccaggt gtttagaaga attggctagt 300

attgaggttc ctgactcaac caacaagttt cggaagatcg agaagaaaat ccaaattcac 360

aacacaaggt atggagaact gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg 420

ggatcatctt ggtctaataa tgtcccccgg tcagaagagt tcaacagcat ccgtacagat 480

ccggcattct ggtttcactc aaaatggtcc acaactaagt ttgcatggct ccatataaaa 540

cagattcaaa ggcatctgat tgtggcagca agaacaaggt ccgcagccaa caaattggtg 600

acgctgaccc ataaggtagg ccaagtcttt gttactcctg agcttgtcat tgtgacacat 660

acagatgaga acaagttcac gtgtcttacc caggaacttg tgttgatgta tgcagatatg 720

atggagggca gagatatggt caacataata tcatccacgg cggcacatct caggagccta 780

tcagagaaaa ttgatgacat tctgcggtta gtagatgccc tggcaaaaga tctgggtaat 840

caagtctacg atgttgtagc actcatggag ggatttgcat acggcgccgt ccagctgctt 900

gagccgtcag gtacattcgc aggggatttc ttcgcattca acctgcagga gctcaaagac 960

actttgatcg gcctccttcc taaggatata gcagaatctg tgactcacgc aatagccact 1020

gtattctctg gcttagaaca aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg 1080

ggccacccat tacttgagtc ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca 1140

ccaaaaatgg tagactttga tatgatcctc caggtattgt ctttctttaa aggaacaatc 1200

atcaacggat acagaaagaa gaatgcaggt gtttggccac gtgtcaaagt agaaacgata 1260

tacgggaagg tcattgggca gctacacgct gattcagcgg agatttcaca cgatatcatg 1320

ttgagagagt acaagagttt atctgcgctt gaattcgagc catgtataga atacgaccct 1380

atcaccaatc tgagcatgtt tctaaaagac aaggcgatcg cacacccgaa agacaactgg 1440

ctcgccgcgt ttaggcgaaa ccttctctct gaggaccaga agaaacatgt aaaggaggca 1500

acctctacta accgtctctt gatagagttc ttagagtcaa atgattttga tccatataag 1560

gagatggaat atctgacgac ccttgagtac ctaagagatg acaatgtggc agtatcatac 1620

tcgctcaagg agaaggaagt gaaggttaat gggcggattt ttgctaagct aacaaagaaa 1680

ttaaggaact gtcaagtgat ggcggaaggg atcttagctg accagattgc acctttcttt 1740

caagggaatg gggtcattca ggatagcata tctttaacca agagtatgct agcgatgagt 1800

caattgtctt tcaacagcaa taagaaacgt atcactgact gcaaagaaag agtagcctca 1860

aaccgcaatc acgatcaaaa gagcaagaat cgtcggagag ttgccacttt tataacgact 1920

gacctgcaaa agtactgtct taattggaga tatcagacaa tcaaactgtt cgctcatgcc 1980

atcaatcagc tgatgggctt acctcacttc ttcgaatgga ttcatctaag actaatggat 2040

actacgatgt ttgtaggaga ccctttcaat cccccaagtg acccaactga ctgtgatctc 2100

tcaagagtcc caaatgatga catatatatt gtcagtgcta gagggggtat tgagggatta 2160

tgtcagaagc tatggacaat gatctcaatt gctgcaatcc aacttgctgc agcaagatca 2220

cattgtcgcg tcgcctgtat ggtacagggt gacaatcaag taatagctgt aacgagagag 2280

gtaaggtcag atgactcccc ggaaatggtg ttaacacaat tgcatcaagc cagtgataat 2340

ttcttcaagg aattgattca tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa 2400

acaatcagat cagacacatt cttcatatac agcaaacgaa tattcaaaga tggagcaata 2460

ctcagtcaag tcctcaaaaa ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa 2520

aacaccgtaa tgtcctgtgc caacattgca tctactatag cacggctgtg cgagaacggg 2580

cttccaaagg atttctgtta ttacttaaac tacctgatga gttgcgtgca gacatacttt 2640

gattctgagt tttccatcac taacagctcg caccccgatt ctaaccagtc gtggattgaa 2700

gacatctctt ttgtgcactc atatgtcctg acccctgccc agctaggggg actgagcaac 2760

ctccaatact caaggctcta cacgaggaac atcggtgacc cgggaactac tgcttttgca 2820

gagatcaagc gattagaagc agtggggtta ctaagttcta gtattatgac taacatctta 2880

actaggccgc ctggaaatgg agattgggcc agtctgtgta acgaccctta ctctttcaat 2940

tttgagactg tcgcgagtcc aaatattgtc cttaagaaac atacacaaag agtcctattt 3000

gaaacttgtt caaatccctt attatctggc gtgcatacag aggataatga ggcagaagag 3060

aaggcgttgg ctgaattttt actcaatcaa gaagtaattc atccacgtgt cgcacatgct 3120

atcatggaag caagctctat aggtaggagg aagcagattc aagggcttgt tgacacaaca 3180

aacaccgtaa tcaagattgc attgactagg aggccacttg gcatcaagag gctgatgcgg 3240

atagttaact actcgagcat gcatgcaatg ctgtttagag acgatgtttt ctcatctaac 3300

aggtctaacc accccttagt ttcctctaat atgtgttctc tgacgctagc agactatgca 3360

cggaatagaa gctggtcacc attgacgggg ggtagaaaga tactgggtgt atctaatcct 3420

gatactatag aacttgtaga gggtgagatc cttagcgtca gcggaggatg cacaagatgt 3480

gacagcggag atgaacaatt cacttggttc catcttccga gcaatataga actgaccgat 3540

gacaccagca agaatcctcc gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg 3600

agggccgcct cgcttgcgaa aatagctcat atgtcaccac atgtgaaagc tgctctaagg 3660

gcatcatccg tgttgatctg ggcttatgga gacaacgaag taaattggac tgctgctctt 3720

aaaattgcaa gatctcggtg caatataaac tcagagtatc ttcgactatt gtccccctta 3780

cccacagctg ggaatctcca acatagactg gatgacggca taactcagat gacattcacc 3840

cctgcatctc tctacagggt gtcaccttat attcacatat ccaatgattc tcaaaggtta 3900

ttcacggaag aaggagtcaa agagggaaat gtagtttatc agcaaatcat gctcttgggt 3960

ttatctctaa tcgaatcact cttcccgatg acgacaacca ggacatacga tgagatcaca 4020

ttgcacctcc acagtaaatt tagctgctgt atcagggaag caccggttgc agttcctttc 4080

gagttactcg ggatggcacc agaactaagg acagtgacct caaataagtt tatgtatgat 4140

cctagtcctg tatcggaggg tgactttgcg agacttgact tagctatctt taagagttat 4200

gagcttaatc tagaatcata tcccacaata gagctaatga acattctttc aatatccagc 4260

gggaagttaa tcggccagtc tgtggtttct tatgatgaag atacctccat aaagaatgac 4320

gccataatag tgtatgacaa cacccggaat tggatcagcg aagctcagaa ttcagatgtg 4380

gtccgcctat tcgagtatgc agcacttgaa gtgcttctcg actgttctta tcagctctac 4440

tatctgagag taagaggcct agacaatatc gtgttgtata tgagtgactt atataagaat 4500

atgccaggaa ttctactttc caacattgca gctacaatat ctcatcccat cattcattca 4560

agattgcatg cagtaggcct ggtcaatcac gacgggtcac accaacttgc agacacagat 4620

ttcatcgaaa tgtctgcaaa actattagtc tcttgcactc gacgcgtggt ctcaggttta 4680

tatgcaggga ataagtatga tctgctgttc ccgtctgtct tagatgataa cctgagtgag 4740

aagatgcttc agctgatatc tcggttatgc tgcctgtata cggtgctctt tgctacaaca 4800

agagagatcc cgaaaataag aggcttatct gcagaagaga agtgttcagt acttactgag 4860

tacctactgt cagatgctgt gaaaccatta cttagttctg agcaagtgag ctctatcatg 4920

tctcctaaca tagttacgtt cccagctaat ctatattaca tgtctcggaa gagccttaat 4980

ttgattaggg aaagagagga cagggacact atcttggcat tgttgttccc ccaagagcca 5040

ctacttgagt tccccttagt acaagatatt ggcgctcgag tgaaagatcc attcacccga 5100

caacctgcgg cgtttttaca agaattagat ttgagcgctc cagcaaggta tgacgcattt 5160

acacttagtc aggttcattc tgaacacaca tcaccaaatc cggaggacga ctacttagta 5220

cgatacctgt tcagaggaat agggaccgcg tcctcctctt ggtataaggc atctcacctt 5280

ctttctgtac ctgaggtcag atgtgcaagg cacgggaatt ccttatactt ggcagaagga 5340

agcggagcca ttatgagtct tctcgaactg catgtgccgc atgagactat ctattacaat 5400

acgctcttct caaacgagat gaacccccca cagcggcatt tcggaccgac cccaacacag 5460

tttctgaatt cagttgttta taggaatcta caggcggagg taccatgtaa ggatggattt 5520

gtccaggagt tccgtccatt atggagagag aatacagaag aaagcgatct gacctcagat 5580

aaagcagtgg gttacatcac atctgcagtg ccctaccggt ctgtatcatt gctgcactgt 5640

gacattgaga ttcctccagg atccaatcaa agcttactgg atcaactggc taccaatcta 5700

tctctgattg ccatgcattc tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat 5760

gcaatgggat attacttcca tctactcatg aacttgttca ctccgtgttc tacgaaagga 5820

tatattctct ctaatggcta tgcatgtaga ggggatatgg agtgttacct ggtatttgtc 5880

atgggctatc gaggtgggcc tacatttgta catgaggtag tgaggatggc aaaaactcta 5940

gtgcagcggc acggtacact tttgtccaaa tcagatgaga tcacactgac taggttattt 6000

acctcacagc ggcagcgtgt aacagacatc ctatccagtc ctttaccgag actaataaag 6060

ttcttgagaa agaatatcga tactgcgcta attgaagccg ggggacaacc cgtccgtcca 6120

ttctgtgcag agagcttggt gaggacacta gcggacacaa ctcagatgac ccagatcatc 6180

gctagtcaca ttgacacagt cattcgatct gtgatctaca tggaggctga gggtgatctc 6240

gccgacacag tgttcttatt taccccctac aatctctcta cagacggtaa aaagagaaca 6300

tcacttaaac agtgcacaag gcagatctta gaggtcacaa tattgggtct tagagttgaa 6360

aatctcaata aagtaggtga tgtagtcagt ctagtactta aaggtatgat ttctctggag 6420

gacctgatcc ctctaagaac atacttgaag cgtagtacct gccctaagta tttgaagtct 6480

gttctaggta ttactaaact caaagaaatg tttacagaca cctctttatt atacttgact 6540

cgtgctcaac aaaaattcta catgaaaact ataggcaacg cagtcaaggg atactacagt 6600

aactgtgact cttaa 6615

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P1 primer

<400> 8

accaaacaga gaatccgtga g 21

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P2 primer

<400> 9

agatgagggg gttcgtttgt ag 22

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> P4 primer

<400> 10

cgcgccgtcg cagtgacc 18

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> P6 primer

<400> 11

cgtttcccac cgaaccgc 18

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> P10 primer

<400> 12

accgacccca acacagttcc 20

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> P12 primer

<400> 13

accaaacaaa gatttggtg 19

Claims (5)

1. A method for preparing a recombinant vector in which the full-length gene sequence of the Newcastle disease virus KU001 strain (Accession No .: KCTC18794P) is inserted, comprising the following steps:

   Synthesizing cDNA using RNA extracted from Newcastle disease virus;

   The nucleotide sequence encoding the NP protein, the base sequence encoding the P protein, the base sequence encoding the M protein, the base sequence encoding the F protein, the base sequence encoding the HN protein, and the base sequence encoding the L protein are sequentially Inserting into the plasmid;

   Including,

   The recombinant vector is made of the nucleotide sequence of SEQ ID NO: 1, the method of producing a recombinant vector.
2. According to claim 1,

   The Newcastle disease virus KU001 strain is isolated from domestic wild ducks, a method for producing a recombinant vector.
3. According to claim 1,

   The nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence encoding the M protein is SEQ ID NO: 4 Consisting of the nucleotide sequence of, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, encoding the L protein The nucleotide sequence to be made of a nucleotide sequence of SEQ ID NO: 7, a method of producing a recombinant vector.
4. A recombinant vector comprising the full-length gene sequence of Newcastle disease virus KU001 strain (Accession No .: KCTC18794P) and consisting of the nucleotide sequence of SEQ ID NO: 1.
5. The method of claim 4,

   The recombinant vector is a plasmid vector, a recombinant vector.

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