CN108660118B

CN108660118B - Novel duck reovirus causing duck arthritis and application thereof

Info

Publication number: CN108660118B
Application number: CN201810494573.2A
Authority: CN
Inventors: 唐熠; 刁有祥; 王鸿志
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2020-12-01
Anticipated expiration: 2038-05-22
Also published as: CN108660118A

Abstract

The invention discloses a duck reovirus, which is preserved in China center for type culture Collection in 2018, 4 and 26 months, and the preservation numbers are as follows: CCTCC NO: v201818. The duck reovirus provided by the invention has good immunogenicity on the duck reovirus which is currently popular and mainly has tarsal joint swelling. The inactivated vaccine prepared by the duck reovirus strain has good safety, the protection rate reaches 100 percent, and the inactivated vaccine can provide complete protection for newly separated variant strains.

Description

Novel duck reovirus causing duck arthritis and application thereof

Technical Field

The invention relates to the field of separation and application of avian reovirus strains, in particular to a novel duck reovirus causing duck arthritis and application thereof.

Background

Avian reovirus (Avianreovirus, ARV for short) belongs to reoviridae (reoviridae) and Orthoreovirus (ortho virus), and can cause various diseases of poultry, and the clinical manifestations of the virus are different due to different virus strains, virulence or infected hosts. Currently, the primary means of controlling the incidence of ARV in avian populations is to control classical avian reovirus infection by immunization using live attenuated or whole inactivated virus vaccines. However, because the avian reovirus is an RNA virus and has a plurality of segments, different strains have certain differences in the aspects of antigen structure, pathogenicity, cell culture characteristics, host specificity and the like. The vaccine prepared by the traditional strain can not well prevent and control the current epidemic avian reovirus infection because the vaccine is easy to generate variation during genetic evolution.

In 2016, the ducks in Shandong, Hebei, Henan and Jiangsu provinces in China have wide-range outbreaks of infectious diseases with tarsal joint swelling as a main symptom, the disease mainly causes tarsal joint swelling of the ducks at various ages, and the tarsal joints often contain a small amount of yellow or blood sample exudates; in severe cases, there is a cheese-like exudate in the joint cavity, with varying degrees of lameness occurring in the affected ducks. The disease mainly causes the reduction of the egg laying of breeding ducks and the weight of meat ducks, the feed-meat ratio is increased, the slaughtering qualification rate of the meat ducks is obviously reduced, and the serious economic loss is caused to the breeding industry of the meat ducks and the breeding ducks. The use of commercial duck reovirus vaccines on the market has not been effective in controlling the prevalence of the disease, suggesting that the disease may be caused by a novel reovirus. Therefore, starting from pathogeny, the molecular pathogenesis and the virus variation mechanism are further researched, and the development of corresponding vaccines is the key point for preventing and controlling the disease.

Disclosure of Invention

In view of the prior art, the invention aims to provide a novel duck reovirus causing duck arthritis and application thereof.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, a duck reovirus is provided, which is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 and 26 months, and has the address: the preservation numbers of Wuhan university, Wuhan university in China are as follows: CCTCC NO: v201818.

In a second aspect of the invention, the application of the duck reovirus in preparing a vaccine for preventing duck reovirus is provided; the duck reosis is mainly characterized by tarsal joint swelling of ducks.

Preferably, the vaccine is an inactivated vaccine or an attenuated live vaccine; more preferably, the vaccine is an inactivated vaccine.

In a third aspect of the invention, the application of the duck reovirus in preparing a yolk antibody for treating duck reovirus is provided; the duck reosis is mainly characterized by tarsal joint swelling of ducks.

The invention has the beneficial effects that:

the novel duck reovirus provided by the invention has good immunogenicity on the duck reovirus which is currently popular and mainly has tarsal joint swelling. The inactivated vaccine prepared by the novel duck reovirus strain has good safety, the protection rate reaches 100%, and the completely-protected duck reovirus variant strain can be provided.

Drawings

FIG. 1: cytopathic effects of the virus isolates of the invention on LMH cells; wherein, A: LMH cells in a normal growth state; b: CPE of N-DAV-SD16 on LMH cells.

FIG. 2: PCR identification results; wherein M is Marker; lane 1 is the sample to be tested; lane 2 is a blank control.

FIG. 3: the homology comparison result of the L1 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 4: the homology comparison result of the L2 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 5: the homology comparison result of the L3 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 6: homology comparison results of M1 gene in virus genome of virus isolate N-DAV-SD16 of the invention.

FIG. 7: homology comparison results of M2 gene in virus genome of virus isolate N-DAV-SD16 of the invention.

FIG. 8: homology comparison results of M3 gene in virus genome of virus isolate N-DAV-SD16 of the invention.

FIG. 9: the homology comparison result of the S1 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 10: the homology comparison result of the S2 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 11: the homology comparison result of the S3 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

FIG. 12: the homology comparison result of the S4 gene in the virus genome of the virus isolate N-DAV-SD16 of the invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, since the avian reovirus is an RNA virus and has a plurality of segments, there are certain differences among different strains in terms of antigenic structure, pathogenicity, cell culture characteristics, host specificity, and the like. The vaccine prepared by the traditional strain can not well prevent and control the current epidemic avian reovirus infection because the vaccine is easy to generate variation during genetic evolution. In 2016, the duck in Shandong, Hebei, Henan and Jiangsu provinces in China outbreaks infectious diseases with tarsal joint swelling as the main symptom in a large range. The symptoms of the infectious disease are different from the symptoms caused by the existing duck reovirus, the tarsal joints of sick ducks are swollen, and the commercial duck reovirus vaccine sold in the market is adopted for preventing and controlling, so that the infectious disease cannot be well prevented and controlled. It is assumed that the infectious disease may be caused by a novel reovirus.

The inventor of the application separates a strain of duck reovirus N-DAV-SD16 from tendon tissues with swelling disease of ducks, the avian reovirus gene consists of 10 segmented gene segments (including L1-L3, M1-M3 and S1-S4), the invention carries out whole genome sequencing on the newly separated duck reovirus N-DAV-SD16, and respectively carries out sequence comparison and homology analysis on the 10 gene segments and the currently reported avian reovirus, and as a result, the L1, L2, L3, M1, M2, S3 and S4 in the 10 gene segments of the newly separated strain N-DAV-SD16 are all positioned in a relatively independent branch and have lower homology with the currently uploaded avian reovirus in Genbank, which indicates that the newly separated strain N-DAV-SD16 is different from other avian reoviruses, the duck reovirus found in the invention can be considered to have larger variation with the duck reovirus reported in the prior art, and is 1 single species of the orthoreovirus genus. Through further comparison, the S1 gene segment of the broiler-derived reovirus causing broiler tarsal joint swelling is recombined in the genome of the novel duck reovirus N-DAV-SD16, so that the novel duck reovirus has important significance for research on the novel duck reovirus.

Compared with the existing avian reovirus, 10 gene segments in the whole genome of the duck reovirus N-DAV-SD16 are subjected to gene recombination and mutation, so that the immunogenicity of the duck reovirus N-DAV-SD16 is enhanced, and the duck reovirus N-DAV-SD16 can be prepared into a vaccine to realize effective prevention and treatment of the existing epidemic duck reovirus.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.

The RNA extraction kit is purchased from Beijing Kangji (century Co.), the common agarose gel recovery kit is purchased from Beijing Quanjin Co., the reverse transcription kit is purchased from Baozhi (Dalian) Co., Ltd., the 2 XEs Taq Mastermix is purchased from Beijing Kangji (century Co., Ltd.), the DL2000Marker is purchased from Baozhi (Dalian) Co., Ltd., the fetal bovine serum and the DMEM medium are purchased from Israel BI, and the PBS is purchased from Beijing Solebao.

Example 1: isolation and identification of duck reovirus strains

1. Virus separation:

(1) clinically, identifying the duck with arthritis disease as positive through PCR reaction, collecting swollen tendon tissues through a autopsy, placing the swollen tendon tissues into a 15mL centrifuge tube, adding a serum-free DMEM medium with the volume 5 times that of the swollen tendon tissues, performing repeated freeze thawing for 3 times after homogenizing, and performing freeze thawing for the next time after shaking for 1-2min on an oscillator after each thawing; centrifuging 15mL centrifuge tubes filled with samples in a centrifuge at 4000rpm for 5min after freeze thawing; taking the supernatant, and filtering with a 0.22 mu m microporous filter membrane for later use;

(2) according to the proliferation characteristics of reovirus, chicken liver cancer cells (LMH) are selected for virus isolation. According to the conventional cell culture method, the culture medium is 25cm²When the cells in the cell bottle were confluent in a monolayer, the medium in the bottle was aspirated and the cells were washed with PBS 2 times, 0.5ml of the freeze-thawed supernatant of the sample filtered in step (1) was added, and the cells were placed at 37 ℃ with 5% CO₂The culture box with the concentration is subjected to induction for 30min, a DMEM medium containing 2% fetal bovine serum is added after the induction is finished, and the time of cytopathic effect is observed and recorded. If no cell lesion appears in the 1 st generation, continuously separating the culture harvested in the 1 st generation according to the method in the step (1) after freezing and thawing until a stable strain is obtained; if no cell lesion exists after the passage 5, the virus is considered to be negative for virus isolation. Finally obtaining a stable strain which has obvious cytopathy 3 days after LMH cell inoculation, namely showing thatThe cells become round, fuse, present a mass shedding lesion (FIG. 1), the blank control cells of the same batch were normal, and the strain was named N-DAV-SD 16.

2. Virus identification:

(1) PCR identification

1) RNA extraction:

extracting virus RNA from virus liquid of the strain N-DAV-SD16 according to the instruction of the RNA extraction kit, and storing at-20 ℃ for later use;

2) obtaining cDNA through reverse transcription:

the reverse transcription kit adopts PrimeScript RT Master Mix with the product number of RR036A from Baozo (Dalian) Co., Ltd, 5 XPrimeScript RT Master Mix X2. mu.L and Total RNA-2. mu.L of the sample to be detected extracted in the step 1) are sequentially added into a 200. mu.LPCR reaction tube, and RNase FreedH is used₂O was supplemented to 10. mu.l system. Placing in a PCR instrument for reaction at 37 deg.C for 15 min; after 5s at 85 ℃ the cells were stored at 4 ℃.

3) And (3) PCR amplification:

amplification was performed using a 20 μ L system: template cDNA × 2 μ L, upstream and downstream primers × 1 μ L, 2 × Es Taq MasterMix × 10 μ L, using ddH₂O was supplemented to 20. mu.L system. Mixing, instantly separating, reacting in a PCR instrument at 95 deg.C for 5min, then at 95 deg.C for 45s, 56 deg.C for 30s, at 72 deg.C for 30s, and storing at 4 deg.C after 10min at 72 deg.C.

An upstream primer: 5'-TGA GAC GCC TGA CTA CGA TT-3' (SEQ ID NO. 1);

a downstream primer: 5'-ATG CTT GGA GTG AGA CGA CT-3' (SEQ ID NO. 2).

4) And (3) PCR identification result:

the product amplified by PCR with the primers showed a specific band corresponding to the expected size of 513bp after electrophoresis in 1% agarose gel (FIG. 2). Indicating the presence of the novel reovirus in the cell culture of each isolate. In addition, other virus pollution is not detected by the conventional duck source virus detection of the isolated strain.

(2) And (3) sequence determination:

the whole genome sequence of the strain N-DAV-SD16 is determined by adopting a second-generation sequencing technology, and the sequences of 10 gene segments of L1, L2, L3, M1, M2, M3, S1, S2, S3 and S4 in the whole genome sequence are respectively shown as SEQ ID NO.3-SEQ ID NO. 12. Homology alignment with avian reovirus sequences published in the prior art is then carried out, and the results of homology alignment of 10 gene segments of strain N-DAV-SD16 are shown in fig. 3-12, respectively.

As can be seen from the figure, the 10 gene segments of the strain N-DAV-SD16 are mostly located in a relatively independent branch, which indicates that the newly isolated strain N-DAV-SD16 is 1 single species of the genus orthoreovirus, unlike other avian reoviruses.

The isolated strain N-DAV-SD16 was identified as duck reovirus. And the strain is subjected to biological preservation, and the preservation information is as follows:

the strain name is as follows: duck reovirus

And (3) classification and naming: duck reovirus N-DAV-SD16 strain

The preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

Address: wuhan university of Wuhan, China

The preservation date is as follows: 2018.4.26

Registration number of the preservation center: CCTCC NO: v201818.

Example 2: TCID₅₀And (3) measuring the titer:

1. the test method comprises the following steps:

strain N-DAV-SD16 passage 5 cell culture (DMEM medium containing 2% fetal bovine serum) was diluted in multiple proportions (10)^-1-10^-6). After the LMH cells of the 96-well cell culture plate are fully paved in each well, 25 mu L of virus liquid with each dilution gradient is sequentially added in sequence, 25 mu L of DMEM medium containing 2% fetal calf serum is added in the last two wells as blank control, the blank control is carried out on the same cell plate for 8 times of repetition, the cell plate is placed at 37 ℃ and contains 5% CO₂After the culture box is used for induction for 30min, 100 mu L of DMEM medium containing 2% fetal calf serum is added into each hole for continuous culture and observation, the condition of cytopathic effect is observed and recorded every day, and the result is judged and read from the 5 th day. Calculation of isolate TCID by Reed-Muench method₅₀The potency.

2. And (3) test results:

the TCID of strain N-DAV-SD16 was determined₅₀The titer measurement results are as follows: 10^-4/0.2mL。

Example 3: animal regression test

40 healthy 1-day-old ducklings which do not carry novel reoviruses and virus antibodies are taken and are subjected to pathogen and antibody detection, and are randomly and equally divided into 2 groups of 20 ducklings. Wherein the 1 st group is an experimental group, the experimental group adopts strain N-DAV-SD16 5 th generation cell culture to inoculate through a paw pad, 0.5 mL/mouse, the 2 nd group is a control group, each paw pad is inoculated with DMEM culture medium as a control, and isolated feeding is carried out. After inoculation, the mental status and the swollen tarsal joints of each group of animals were observed daily and recorded.

The experimental group showed swollen tarsal joints lesions 7 days after virus inoculation, and 20 experimental groups showed swollen tarsal joints lesions 9 days after virus inoculation, which showed the same symptoms as those of natural infection cases and showed good health conditions of the control group. And (3) carrying out pathogen re-separation on the tarsal joints with the pathological changes of the experimental group according to the pathogen separation operation, and separating to obtain the novel reovirus.

Example 4: preparation of inactivated vaccine

(1) Propagation and harvesting of the virus:

and inoculating the separated and identified novel duck reovirus N-DAV-SD16 into a well-grown LMH cell line, and carrying out mass propagation on the virus to obtain enough virus liquid. Freezing the obtained sufficient cell virus liquid at-20 ℃, freezing and thawing twice, and collecting the cell virus liquid.

(2) And (3) virus purification:

and detecting whether the obtained virus liquid contains other common viruses or not by using established detection methods such as PCR, RT-PCR and the like. The detection items comprise Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Parvovirus (DPV), Goose Parvovirus (GPV), Infectious Bronchitis Virus (IBV), Infectious Bursal Disease Virus (IBDV) and the like, and the seed virus is purified.

(3) Inactivation of virus liquid:

and inactivating the qualified virus liquid by using formaldehyde, wherein the optimal inactivation condition is to add the formaldehyde with the final concentration of 0.2 percent and stir and inactivate for 16 hours at 37 ℃.

(4) Preparation of the vaccine:

preparing an oil phase: taking No. 10 medicinal white oil, Aluminum stearate (Aluminum tristearate) and Span-80 (Span-80) according to a proportion of 94: 2: 6, mixing, stirring uniformly, and sterilizing at high temperature and high pressure.

Preparing a water phase: adding Tween-80 (Tween-80) into the inactivated antigen solution, and shaking and mixing to completely dissolve Tween-80, wherein the final concentration of Tween-80 in the water phase is 2-4%.

③ emulsifying: oil phase and water phase the ratio of 2: 1, adding 2 parts of oil phase into a tissue homogenizer in a super clean bench, slowly stirring 1 part of water phase during the stirring, mixing at 6000rpm after the water phase is completely added for 10min, emulsifying at 8000r/min for 20min, and subpackaging for later use to prepare the inactivated vaccine.

Example 5: quality test of inactivated vaccine

The inactivated vaccine prepared in example 4 was subjected to a procedure comprising: quality inspection of dosage form, centrifugal stability, viscosity, sterility and shelf life is carried out by referring to pharmacopoeia of the people's republic of China (2015 edition).

The results were: the preparation form of the inactivated vaccine prepared by the invention is water-in-oil (W/O); the centrifugal stability, viscosity and sterility test were in accordance with the regulations of the pharmacopoeia of the people's republic of China (2015 edition).

Example 6: safety test of inactivated vaccine

20 ducklings of 1 day old are taken and divided into 2 groups at random, and each group comprises 10 ducklings. The 1 st group is an experimental group, 0.5mL of the inactivated vaccine prepared in the example 4 is injected into leg muscles, the 2 nd group is a control group, the equivalent amount of the sterilized white oil adjuvant is injected into the leg muscles, after inoculation, the mental states of animals in each group are observed every day, whether local inflammatory reactions such as red swelling, thermal pain and the like occur at injection positions or not is continuously observed for 2 weeks, after 2 weeks, the experimental animals are dissected, and the vaccine absorption condition at the injection positions is observed.

As a result: the experimental group shows transient mental depression but recovers quickly, the experimental group and the control group are observed for two weeks continuously, the growth and the development of the experimental group and the control group are normal, the mental state is good, and the vaccine at the injection part is found to be well absorbed by a caesarean examination experimental group without inflammatory reactions such as red swelling, tissue necrosis and the like. The results prove that the trial vaccine is safe and harmless and has no influence on the growth of animals.

Example 7: protective testing of inactivated vaccines

60 ducklings of 1 day age are taken and divided into 2 groups at random, and each group contains 30 ducklings. Wherein the group 1 is an immunization group, the inactivated vaccine prepared in the embodiment 4 is injected into leg muscles, 0.5mL is used, the group 2 is a control group, the equivalent amount of sterilization white oil adjuvant is injected into the leg muscles, 0.2mL of novel duck reovirus liquid is injected into the legs of two groups of ducklings 10 days after immunization, the ducklings are isolated and fed in groups, and the mental status and the death status of the two groups of ducklings are observed.

As a result: the immune group has transient mental depression, but does not show clinical symptoms of duck reovirus disease and death; the control group of ducklings have obvious clinical symptoms (tarsal joint swelling) after being attacked by toxin, the number of the ducklings died is 8, and the result shows that the immune protection rate of the inactivated vaccine can reach 100%.

Example 8: determination of duration of immunization for inactivated vaccines

20 ducklings of 1 day old are taken and divided into 2 groups at random, and each group comprises 10 ducklings. Wherein the group 1 is an immunization group, and each leg is injected intramuscularly with 0.5 mL/mouse of the inactivated vaccine prepared in example 4; group 2 is a control group, and the leg is injected with the same amount of sterilized white oil adjuvant; after immunization, serum was collected every two days and antibody was detected by indirect ELISA.

As a result, the antibody level in the immunized group was found to start to rise significantly at day 4, peak at day 10, and slightly decline but still maintain a higher level within 30 days thereafter; the antibody levels of the control group were negative throughout the experiment. The inactivated vaccine disclosed by the invention can quickly reach the antibody level with high concentration in a short time, has long immune duration and can provide quick and long-term immune protection for the ducklings.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

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<120> a novel duck reovirus causing duck arthritis and application thereof

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<400> 4

gctttttcct caccatgcat gtcaacgggt ttgatgatgc tactctctcc tacgcacagt 60

ccatatcggg ggttatccca atgacaaata agttatttga gcaagcatct acctcaatac 120

gtgccctacc acgctcacat gtttatgcta tattagataa tgttaatttt tcagtttcat 180

gtgtcatccc gaatcgtatt ttccatcatc ctgatcactc tgagtacttt tacattgatg 240

cggttaacag agttagacgg aaacaagtta tcgatcctga tgatgtattt gtgccaaact 300

gcaatttgca gggtcttatt actccaatgg agagattacc aaattatggt caattgtctg 360

agactatttc gtcgaacgct cgggatggct tgccatctgc acgcgtagca gctacttttt 420

ataacacttc ggtgtcacag gctcgccaag ttaaagctcc acttgaatcc tttttgttgc 480

ctttgctatt atctgagact tgctcattat cagacgatcc ttgcggactt gacaccgcag 540

cttctcctcc gatccatacc aatctggcgt tatgggtgtt acgcgaaatt agtcgaacta 600

tttgtggatc ttcgaatgac cgttcacctt ggttactgct cgattcaggg gttgcgtggt 660

tcatgtctcc gttaatgtca tcggccattc cacctcttat ggctgactta acgaatctag 720

cgatctacaa acaaatttgt tctgtgtctg atgagcttca ttcccttgca gttcaagtgg 780

ttttgcaggc cgcggcgtca caatcgtatg gacactacat attgcagacg aagtcagtat 840

tcccccaaaa tacgttacat aacatgtttc gtacgctcac tgatggtatt gttccggtca 900

tagattggtt agaaccgcgt tcaaactatc gcttcatgct tcaaggtgcg cgtaaagtga 960

cttcagatga tgcgaatcag gctccggata acacggaagc cgcggagcaa ctcggtcgca 1020

agatggggtg cctcgatgtc gtacgctcct tacgtaagat gtcctcgtcc atcactgtcc 1080

attcacacga tgccatgacc ttcgtacgtg atgctatgtc gtgcactagt ggcatcttta 1140

tcacgcgtca acctactgag accgttttaa aagagtacac tcaagcgccg accattgaag 1200

tccctattcc tcaatcggac tggtcaccgc ctattggatc tctgcgatat ctctctgatg 1260

cctgttccct ccccgctgtg tatctggcta gggcttggcg cagagctgcc tctgctgtag 1320

tagataatcc gcacacttgg gaccctttat atcaggccat ccttcgctct caatatgtga 1380

cgtcacgcgg tgggtccggt gccgcgttaa gagatgcttt gaaggctgcg gaagttgagc 1440

ttcctcagta tcctggggtc agtgttaaag tggcgaccaa gatttatcaa gcggctcaga 1500

ctgctgacgt gcctttcgat aaattatctc gtgctgttct agctccattg tcgatgggct 1560

tacgtaacca agttcagcga cgtccaagaa ccatcatgcc catgaatgtg gttcaacagc 1620

agatttcagc ggctcatact ctctccgctg actacattaa ttatcacatg aacttgtcga 1680

cgacctcggg tagcgctgtg attgagaagg tggttccatt aggtatgtat gcatcctgtc 1740

ctcccgctca agcggttaat attgatatta aggcttgtga cgcgtccatc acgtaccagt 1800

attttctttc tgttatcgtc ggtgccattc atgagggtgc agcaggccgt cgtgtctcgt 1860

cttcattcat gggcgttcca ccaagcgtgc tgtccgttgt cgatgctagc ggagtgactt 1920

catccatgcc catctcaggt tttcaagtca tgtgtcaatg gttggctaaa ctttaccagc 1980

gaggttttga gtatcaagtg acggatacat tctcacctgg caatattttc acgcatcaca 2040

ctactacttt tccctctggt tcaacagcga cgtctacaga acatactgcc aataatagca 2100

cgatgatgga tggcttcctg cgcgcttgga ttccttcctc cggtgcgtcc gacgtactga 2160

agaagttctg caaatccatc tcaatacaac ggaattacgt ttgtcagggt gacgatggtc 2220

taatggtcgt tgatgggcta tcgacaggta aattatcagg cgagataatc gatgaatttg 2280

ttaaggaatt gagagcctat ggtaaatcat ttgggtggaa ctatgatata gagtttaccg 2340

gaaatgcgga gtatctaaag ttgtatttcc taaacggttg ccgtatacct aatgtttctc 2400

gacatccgat ctgtggcaaa gagcgcgctt caggggacaa gttagaaatg tggccgtcca 2460

ccattgacat cttcaatggc atatttgtga atggtgtgca tgatggtttg ccgtggcgca 2520

gatggttacg ttattgttgg gctcttgctc ttatgtattc tggaaagacc gtgcgtcatg 2580

atgattctga ggtgttgatc caatatccta tgtggtcttt tgtgtattgg ggtttgcctc 2640

ccgtgagcgc gtttgggtct gatccatgga tcttttctcc atatatgccc actggtgatc 2700

atggtttcta ttcaatgttg actttagtgc gccctctgat cactaacttg tccccatcgt 2760

cagacgcttc gggattattt ggtcaatgcg atcacaacgt tttgttcaat tctgagctag 2820

tttatcaggg ctattacatg gctcaatgcc cacggcaacc ttctcgctcg aaccgtagag 2880

atgatcccga ctctgtacag cgcttcgtca aggccttgga gtcttacctt tacatttccc 2940

ctgagctaaa atcgcgagtg cgccttggtc gtgaccgctg gcagaagttg gttgggtata 3000

cggaaaaatc tcccccgtcg cttgatgacg tggcgttcaa atggttccgt agtgcacagg 3060

aagctgatct cccaaccgct acagagattc aaagcatgga tctggccttg ctggcagcca 3120

gacgtaggac atatcagggc ttctccaagt tgttgaatac ctatttaagg gtaacctggg 3180

atttatctga tcctgttgaa cacgctgtag atccccgcgt acccttgtgt gccggtgtct 3240

ctccatcaaa tagcgagccg ttcctcaaat tgtactccgt aggtcctatg atgcaatcca 3300

cgcgtaaata ctttagcaat acgctattta tccatcggac tgtgtctggt cttgacgtcg 3360

atgtcgtcga tcgtgcgctc cttaggttgc gtgcccttaa tgcgcctgat gacgtggttg 3420

tagctcaact tttgatggta gggttgtctg aagccgaagc cgctacatta gcagcgaaaa 3480

tacggacgat ggatatcaat gccgtgcaat tggccagagt tgtcaactta tccatccctg 3540

actcatggat gaccatggat tttgatcgct tgatacgaga tatcgtgtct gtcactcctc 3600

tgaccgttcg atccctaacc accgatctac cctctggcgt accgtgggct cgcgcgatct 3660

tacagttctt aggtgcgggt gtcgccatga cggccgtcgg acccttgcgt cgtccctact 3720

tacactcagt tgccggaggc atgtcttcat tcattaagca gttccgccgg tggatgcgtg 3780

ccgaaacgag gtagcgtccg tgcccggcat ggctcgagga attactcatc 3830

<210> 5

<211> 3876

<212> DNA

<213> L3 Gene fragment

<400> 5

gcttttccac ccatggctca gattcgaggc cttcggttgt ctacgacgct ctcagctcca 60

cctccacgca agattataac atctcacact tatgatgagc tgatctccgc tctgaagtta 120

gcaaccaagc cttggcgccc tttgaagtcg cgaaataatg attctgtcac ggcagtgcag 180

ctcctttttc cccttaatgg ttatattgaa cccatgttca tgttggaaaa ggatatgacc 240

cttagtgatt ttgaggcctg gttgacgcct cttctatctg cactcgctga ccagttgctt 300

agacactacc ctatcgccgc ctatcacggg cgtttgatta atccgctgct atctaatgca 360

attgttgccg ctttcttgtc taacgtgcct tatgcgcatg cattggatca tctcttcctt 420

gttagaggaa acgtcgagga tattatggat gcggggatcg caattcagaa tcacttgtgg 480

ttcgatcgcg gtgcactagt gacccctgct ggacagaaat ttgttcagct gactggctat 540

aacttctcct ctaatgatcc gtgtctattt tctaagcaat tgcgttgtta tggcctcgtt 600

tactactttc tcgacatggc cgaatgtctg gcgtattgtt ggcgtcatct atccaattca 660

actccactga tacactttga ccgtccgtcc aatggagttc attgtttggt gccctctgaa 720

tccacgacgc ctatcgctgg ttcgttacca gtgtcagcac tcagctctat tttgttggaa 780

tcctgtcttc agcaatctac aattaatgcc cttactccca ctggttcgcc cgtcattaga 840

caaatagaag cattgttgcc tatatcatca ccgtttttcg aacgacggaa cactctggaa 900

tattctctct tcgcactgtc aaatgctctg gtaaatggtt atcagcttgt agacttgcgt 960

tccggccacc ctgattgcgc tactgttgct gccatcctag ctagattgat tgatttctct 1020

aaggatatca ccgttattca accgcgccct gctctctttg ctatcaacag cgacagtccg 1080

cttacgtata gtggagaaaa tgctaatttt atttcgcgct tgacttgtgc gtccggtaga 1140

cctattggtc cggtcgttgt cggtaaatcc gttgatcatg ccgttggttg gatgccccag 1200

tttgaccccg ccacgtccta caaccctgat ctctcgatgg actcacttgc tcgtgccacg 1260

acactgcctc tccgtgctaa gtattcgact ttctggtcag gcccagcatt gttttctttc 1320

gcttcatgta ataggcacaa tggtgtatat gacatacagt tcatggctca atttcctcct 1380

acttacttta gtgatgatga cgccttttct agatcacgat tctcttctta tcgtgcagtt 1440

agggaccggt cattgttgaa ggataccgct aatttgatgt acatctcgaa tttgtccagc 1500

tctcacgacc atcgtcttgt cccagattct aaaactatga tttatgtggg ttcctctggt 1560

actcatgtag ataatcaacc ttctatcatt aaacctctct tagctggaac tcttccgggt 1620

gtttttcgcc ccctttctat aaaacaggtt ggttgggagg tcactaatgg aacgatttgt 1680

gatgttgagc ttcctttagc cactggtacg ttcttcttcg tgtacagtga tgtggatcaa 1740

gtgcaatcag gtgattctga tttggacgcc tcctcgcgtc gcttttgctc ccaattggac 1800

atgctaatga agttgacgtt tactggtgga tcgcttgtcg ttaaatgcaa ttttccgact 1860

agtctagtgt ggcgtcacat cttctccact gtttctcctt atttctcggc tattcattta 1920

atgaagcctc tcgtgtcaaa taatttagaa ctgtatctat tgtttgcgga gcgtttgccc 1980

gttcctgacg tcgcgttccg tccttcagcg gacgttgtcg ttttctggcg atcacagcta 2040

caacgctatc gagtgttgcg tgattccttt tctaatgtgc cctccatcgg gtccactctc 2100

actttagatg aacctttgac tgtctctatt ctcaattttg ttgacgtcac ctccctttct 2160

tctcttgagg atcaacgagc cttatctgct ttttcagttt taacttctct agggtcacag 2220

aaactctcgc ttcatcctta ctttgatagc taccgcacgc agctcactgg aataattact 2280

ccacattcac gtaatcttct agatagactg gcgtacgtcc cgcgcgtttt tccttcgacg 2340

attgatgtgc aacatcgtgt catggcctct tcagatccag aaatttttgg ttttcgttct 2400

aattcatgga ctcaactgtc cttcttctac gacgcgacgt taacttctat tgattttact 2460

gatgtaaagc actggttaga tttagggacc gggcctgagg cgcgaccgtt gtcttttctg 2520

ccttccgatc ttcccgtcac gttatgtgac actcgtccat tcatcttccc ttccggctgc 2580

tgggctactt tcactgattt cttaagttat gactaccttg tcacgaatgt cgtgctctca 2640

actggtgccg atgtcgtatc ctgtgttctt tctctgggtg cggcctgtgc tgatgccaac 2700

ataactttac atgaaggcgt gcggcagctg atttcccaat gcgtggatgc caatgtcaag 2760

acattgttcc tgcagcttaa ttgtcctctc ccatctgcgg gtgatgtatc tcgggagatt 2820

cttgagatgg ttcagactaa ttcaacttac gtgtttcata ccttgggtcg tattgaaccg 2880

ttcattccat actccgctct ttcagagata gttgaggact tgtgtcccgg catcgtcatt 2940

gaaattaaga ctatggatcc ctctctctca tggcttgatt acgctgttca atccaatgcg 3000

tcagtgacgt cggatgatat cgtattggca atgcgtctgt ctcacttctg tccacttttc 3060

gtgtttcatt ttgaccgtca gtctgctcaa tttccggatg atgcgcgtgt tgggactcct 3120

tttactgtca cgctgttaga ttatgaagat actcgttcat acgaggtgac gttagataat 3180

gtcactatcg ccaccgttac cgcaggtgct ttggtgggtt tctcatctgg tgtcagcgtc 3240

agttcaacca acaatcagct tattttaact atcgattccg caagtccagg aattctctcg 3300

gtcattcaga ttcttcccgc tcgtatctct ttaggcagtt gtgtgataga agcaccggat 3360

ccatctctct ccttgatctt ccccgccacg ttagatacct ctttgtcggg aaccgatttg 3420

gagctgtatt tgtctgactg gtacgatgtt gcattatttt acgtcgatga aatccactcc 3480

cgcttgctgc cagtgtccga taccaagtat gaaatatatc gcaaggatca ggcgccgaac 3540

agccgggtga tcaactatat tttcgatcgg tctgatgtgt ttttcaagct agtgttatgt 3600

gacgtatctc cctcaggagt aggccgtttc atctaccgtg agttgccaga attaagttca 3660

cctgtttggc cagacaacgc gcgcactttc ttgtccatac cgttcgagtc acccatggtg 3720

attgtctcgc cggacggacc tgttaattac gatggcgcaa actttactcc tccaaactca 3780

tggttgacgg ttgatggcag tacctgcgtt gtagatggcc gtccttcgtt ctacgtgccc 3840

cctggccgat atggtctggt gagagtctaa acgacc 3876

<210> 6

<211> 2199

<212> DNA

<213> M1 Gene fragment

<400> 6

atggcctatc tagccacacc tgtgctagga gtcggttctc gcattaccgc tttagatcgt 60

actattgatg ctatcacgtt gaaacctcga atcgacttac aagatgtata cacaattgat 120

cccactttga ctctgcgtca gatagagtta atctcttcgg gaacttcaat ggacgatatc 180

gctcgtggac tgttgcaccg agactggcgt cgtcaatcca tcatcgtttt gcttccctcg 240

cgtcgctctc tccttgagta tctattgtct aacccttctg tctgtccaga cggtttagat 300

cgttctcgac ttaaaggatt tcagaagcgt ccaaatgatt ttcgtgttca agatttcttc 360

tctccactga tcacggactc gacgtcaatt gctacatact ctcgatggct taatgcccac 420

cctgttgtgt actcaactac tcataaggtc gctggtgctc gggtgcgtct ctttggacct 480

gccaaattat acattctgtc acctgacgtt cttcgcgaat tatccatttt gagatccacg 540

gatcgtgtcc tcgttgtacc tacagcacgt gtatatgttg gttgctttcc tagcgcttcc 600

actagtaatt gtgtgctcac tgcacgcgac cgctggaatg ctcctgacgt tcatcccgtt 660

gtcaaggcaa tccaattagc atatgaccat caatatcgtg tcaccgctcg ctatctttcg 720

gatccccttg tctccgccct tcttgttggg aatcggtcgg tgaagacctt gaaggtacag 780

ccagtagagg ccagagcagc acgatcagtc ggcattcgcg ttcaagcgat gacgccccct 840

cgtggtatca acacctctat catccaagtc gttgatctca ggctgcaatg tcgacattct 900

ctcattccca ccgaaaggcc cttcccgctg acatttatcg gcctcccatc ctgtttgctc 960

cagcatttgg atttgacgct atctgacgat tgggtgccta ttcgtgatcc cacgggcatg 1020

tttgaaatgt ggttcatgat tcttacgctc acttgtgata agattcttga tggacggggc 1080

aacgctgttt ttctcatccc cagttctact aatgcattgt cgattaatta tgtacagctt 1140

acatcgaccg cgtctcaacg ccctcagtca ttagcggcaa atgcatctgg acggatagat 1200

tctatcggac tatgtatgcc taaggggtct tttaagtcaa ctatgattaa atttctcact 1260

ggcttggaga tttgcggcac acgagtgatg tactcggacg tcgtgatgga cagtgatgac 1320

gtgggtgacg ctttggatcc tacttttgaa acggctttgt atgatgcact ggtagcactt 1380

gatccgcctt ttgacgttga taagttggct agccccactg atctagttaa tcaggagtac 1440

gttgcgtctc atatgtaccc gacattctta cggcttgtca atgagctgct gactcctaag 1500

gcttcagagt tgtactctga gcgtagcgtt gaattccgat ctcttactta cgcgcacgct 1560

gattctgaat ttcttaactc atgctggacc gctcgcttaa tgcgttgctt tatcaactat 1620

catgaagagc agaatatctt acttcgtcct ggacgcgttg gtggggtgtt atttcaagtc 1680

gcgttgagcc gttgctataa gatgttcgct acttccactc ctgcttcccc tctgtcattg 1740

ttcctcaagt cgttgttcgt tccttggatt gagtctgccc cactgttagc gaatctaacg 1800

ccaaatgagt cttctcgtgt gttagcatgg tatattcctt cctcgtactg gagcgacaat 1860

ggttggtgcg tttgtgacac tcatcgtcac gtcaccttct ccttcatccg cggtcttccc 1920

gccgacctgt cggtgttaga tctgtttgat tggtctcgat tccgcgcgac tataaacgtg 1980

gacacgtctc tcgtggagct aggcgcagac attcgtgcgg tcaaagtatc agtccattgg 2040

acatctcaga agcccactgt ggacgttttt gacaatcgtg cgcttttcac ccccttccag 2100

cactaccatt tgagtctcca ctgtaattgc gcacctggtc gacctttctt cgcgaagaac 2160

atgaagctat atttgtcgac ggtaggaggc gagcactga 2199

<210> 7

<211> 2208

<212> DNA

<213> M2 Gene fragment

<400> 7

accaccgaga tctacactct ttccctacac gacgctcttc cgatctcttt ctcacaagat 60

gggtaacgcg acgtctgttg tgcagaactt caatatccaa ggtgatggta atcattttgc 120

tccatctgct gagactactt catccgccgt accgtcatta tctttgaatc ccggactgtt 180

aaatccaggt ggtaaggcgt gggtcctgat tgatccatct ctaaatgctt ccgatccttc 240

atcactacgt ctgatgactt cggctgatct atcaacactt cctcgatctg ctactagtaa 300

ctctaccggg tttctcccca cttctggcat gtatgccatt gctactaagg agacgttgag 360

tgtaattact gagcacgcga tttcccagtt tgataagtta cagatggctt gtgagttgga 420

ccgcgattat ctggatgcta gaggtgtttc tcctgagtct gtgaatattc atagttatat 480

agcctacgtt gattgcttcg tgggtgtatc tgcaaggcag gctgcgtcaa attttaagcg 540

gcatgtgcca gttatcacca aatctcgtat gacacaattt atgacatccg cgcagaatat 600

gttgcaagtg cttgggccct gggaacgtga tgttcgtgag ttactcacta ttcttcctac 660

ttccactacc gctggtaaaa ttacgtgcga catgaagtct gttgtcgctt tcattgatga 720

tcagctctct gataccagtt tgtgtcgtct gtaccccgac tgtgctgctg cggcggtggc 780

tagacgtaat ggtggcattc gatggaagac acctgatact gacgaggctc cttcacttgc 840

aactaacgat attgctgctt caactatggg tacgcttgcg aatactacac cactggctga 900

gaagtcgaac tcgggcgagg agtcgatgcg cttggttagt gatgttggcg tggacatcgt 960

ttgttctcgt ggccccatca gttcttcagt ttggtcccgc acggttgaac ccaaatcgta 1020

caatattaga acccttcgtg tagaagaagc gctttggcta cgcgagtgcc aagcgactac 1080

tggttttgat gtacagtaca cgctgcccga ccagactaca cataagcatt tctggcttca 1140

gaaggggtca gtcgtcataa atcttgagca aacgggtagt atgatgttcg atgtgaacat 1200

agcgggtaaa gattacaaga agggcacctt taatcctgat aatcataaat tggtcctctt 1260

ggttatgcag tcaaagatcc ctttcgagtc ttggaccgtc gcttctcaaa ttactggtat 1320

cgctcaagtg gctgaggtca ctgtgcatgc tgctgatagt tcgactccta accaaaagat 1380

aatcggtgaa acttcgctgt cttatttatt tgagagggag acggtgacca catccaatac 1440

tgaagtcaat acatatctgt tgtgcacttg gcagcttgac gacgcgcaga gcaatgacgc 1500

aaacgcctgg ccagatgctt gggacgggat cacaacattg accccactta cgtccggtac 1560

tgtaaccatc aaggggactt cggtggattc tgtcgtaccg tctgatttag ttggtgctta 1620

tacacctgag gctttggctg ccgcgcttcc taacgacgct gggttaattt tggctaataa 1680

ggcaactaaa ttggctgacg ccatcaataa ggaggatgat tctgtgattg atgagtcttc 1740

tccctttagc acccccattc aaggagttct ggctgttcaa caacttgata ccgtggggac 1800

acgcggtaca cgtgcactcc agcctccatc cattctgaaa cgcatcgcct cacgagctct 1860

tcacatgttt cttggtgatc caaagtctat tctaaaacag gcgacgcccg tattgaggga 1920

ccctgacgtt tggaccggct ttgttcaagg tgttagagac ggcatccgga ctaagtcgct 1980

atccgctgga gtacggtccg tgtataataa cgttaccgcc acacagtctg tacaaacgtg 2040

gaaacagggg ttcctgacga aaatacagac gttgttcaag ccatcgtgag gtgctaaggc 2100

ctctctctgc ggcgggtcgg tgggcacgtc gtagtgacgc tgaatgcacg gggaggtgac 2160

gctccctgga ttggcaagat cggaagagcg tcgtgtaggg aaagagtg 2208

<210> 8

<211> 1996

<212> DNA

<213> M3 Gene fragment

<400> 8

gctttttgag tcctagcgtg gatcatggcg tcaaccaagt ggggagacaa gccgatgtcg 60

ctctcaatgt ctcacgatgg atcatctatc cgcagtgccg cctcacagtt tctgtcggtt 120

cccctgtctc actcaacgcc aatcccacct caacggaaga ccgtattgct gaaattcatg 180

atcggtgatg acctggttac cgtgcagggc gcgctcgctc cttttgatga atactggtac 240

gataaccaac ctctattgtc tcaagctgtt gagctgctcg cctctgagga tcgtctgcgt 300

caattcgagc attatgagaa gtttctactt aagaagggcc accaaattgc tgagatcatg 360

aataggctac gtcttttctt caccgacgtt ctcaaagtga agatggaagc tgatgctctt 420

ccttctctag ctcaatacct gatggctggt actttggatg ctgtctccac cgttcacgaa 480

cctgatgctt gtgttccagt cacttcaaaa atcatagcta agcagcagac tgtgtccaag 540

tcccctggac gtcttgctga agaggagtat aatgttatta gatcacgttt cctcactcat 600

gagatctttg acttaacgtc tgacttgccc ggtgttcaac cattcatgga tatgtactat 660

gccaccgttc ctcgtgccga ttccaccggt tggtgtgtct accgcagaaa aggtctattg 720

attcatgccc ctgatgagca atactcggat ctgactattt tcaccacccg tcttacggca 780

gcgcgtgagt tacagcttgt ggctggggag gtcgttgtgg cttgcttcga tcttatggat 840

atctctgata ttgctccatc tcatcatgca tcggttcaag aggaacgtac tctcggcact 900

agcaagtatt ccaacgttac agctaatgag catccgttgg tattcttttc acccaatgca 960

ttacgctggg caatagatca tgcctgtact gattccttga tttccactag gaatattcgg 1020

gtctgcgtcg gtattgaccc cttagtgacc agatggactc gcgatggcgt gcaggaggct 1080

gcaattctta tggatgacaa gctaccctca gcaggacgtg ctcggatggc tctacgaacc 1140

ttgcttctag cgcgtcgctc accaatgacg tccttcttac taggtgctct caagcagtcc 1200

ggtggtcagc taatggaaca ttatcgatgt gatgcggcta ataggtatgg atctcccacc 1260

attccagttt ctcaccctcc accgtgtcca aaatgtcctg agctgaaaga acagatcacc 1320

aaactttcgt cagctcctgc gcctaaagtt gactcgtccg ctggtcctgc cgtgctgttg 1380

tcgaagatcg ctgagctcca acgtgctaac cgagaactgt ccttgaagtt agtggacgtt 1440

cagccagccc gagaagacca ccttctagct tacctcaacg agcacgtatg tgttaacgct 1500

aaagatcacg agaaaggtct actagcccgt tgtaatgtct cgagtgattc aatcgccgct 1560

atccttggtc aacgtttgaa aaatcgagaa cggtttgaaa cgagactacg gcacgaggct 1620

ggtgcggagt gggagccacg agtggaagcg ttgaatcaag agttggctaa ggcgcgtatt 1680

gagcaacaag atatgatgac tcagtcctta cagtacctga atgaacgtga tgaactgctg 1740

cgcgaggtgg atgagctcaa acgcgaactg gctaccctac ggtttgctaa tgtgagacta 1800

aatgccgata accaccgcat gagtcgtgcg acccgtgttg gagatgtatt cgtcagtgat 1860

gttgatccct taccacccgg tcttcctggt gaatcgaaac catccattga agaactggta 1920

gatgatctgt gagctttgcc ttgtgactcg acttctctct gattccatgt acccacggcg 1980

gactcggcta ttcatc 1996

<210> 9

<211> 1644

<212> DNA

<213> S1 Gene fragment

<400> 9

gctttttcag tctcttgtat cgatgttccg tatgtcttcc ggttcatgca acggcgcgac 60

gtcaatattt ggtaacgtgc attgtcaagc ggcccaaaat accgcaggcg gagatcttca 120

agctacctcg tcattaattg cttattggcc ttatctcgct gctggtggtg gtctcattat 180

aattttaatt attgttatag gtataatctg ttgttgtaag gccaaagtta aggctgacgc 240

taccagaagt gtgttctatc gggagttgct tgctctgaac tcgggcaagt gtaatgcagc 300

acctccgtca tacgacgttt gatgtgcggc ggtttgagtt ttctccgacg gtgtttgaag 360

agtgtttgac tccatctttt accgctgtga ctgacactga ccctgtgagg tactttaata 420

ttgagcttcc gtcaactcac cgtctcctcc cttggcttcc cgttcttctg ttccaatcct 480

gtaaagtgca tgtttcttta gtacgtagat tctctttatg ctcgacttta tctgatattt 540

gtgagtacga ttgcaaattg cttccgtcta ttaacgctat tgtgtcgaat ccagtgtcga 600

gcgcggtttc atctatcgtc gttcactggg atggacggat taactcaaca gcagcgaaga 660

gaagtcgtgg ggttgatact gtcgttgact tcgagcgtga gtataagttc tggcgatttg 720

acgcaaattc gtgagcgtct ttccgctttg gaatctgcga ctgcgtcgct gaacgaatcc 780

gttaatacag ctttgtctag gttagtggat ttgtctgcat cgcttgataa cgtggcggcc 840

tcgttagcgg agacgaaagt ggaaatgaac tcactagttt ctgacgttca gggtttgcga 900

gcttcccttg actcttctgc ttcagagctg gcttctctat cttcattggt gcgtgatcac 960

ggctcttcga ttgctggcct acagagagaa gtaagtgcct tatcgagtga ggtaggcaac 1020

cttaaaacct cggtatcatc gcagggcctt cctatcacta gccttgagaa acgagtgcaa 1080

gctttagaag gtggttctag tacgactctg tcatttgctg atcctcttaa gttagaggct 1140

gggaccgtgt cactcgaggt agatccgtat ttctgctctg tgaatcgtaa tctgacgtcg 1200

tattctgctg atgctcagtt gatgcaattt cagtggtctg tgaaagggga agatggcgcg 1260

gccaactcta ttgatatgga cgtgaacgct cactctcatg gttcacgcac tgattatctg 1320

atgtcaacca agcaatcatt gactgttaca acgtctcccg ctactcttgt ctttgaactg 1380

gataggatta ttgctcttcc ctccgacctt tctcgcctaa ttccatgtta tggttttcag 1440

caagccactt ttcccgttga tatctccttc cagcgagatg gcgtttcgca tacgtatcaa 1500

gtctatggga agtacacatc ttctcgcgtc ttcaagacta cgttctcgcc tggctcctca 1560

ggtcccgcag tgattaagtt tttgaccgtg cgtacgggca tcgataccta aggtgtggcg 1620

ccgtacgggg gctggttatt catc 1644

<210> 10

<211> 1322

<212> DNA

<213> S2 Gene fragment

<400> 10

gggcgcgtgc cgtgtacgac ttcttttcta cgcctttcgg gaatcgtggt ctagcgacga 60

atcgtactca actatcatca ctactatcaa gctcgaattc cccatggcaa cgttttctat 120

catcaatgac tccattgaca gcgccgggca tcgtttcgac acctgaagca ccctatccag 180

gttcgttaat gtatcaagag tctatgctcc acagtgctac cgtccctgga gtacttggca 240

atcgcgacgc ttggcgtacg ttcaatgtct tcggactttc atggactgac gaaggactgt 300

caggactagt ggctacccaa gatcctcctc ccgccgcccc gtatcagcca gcctctgctc 360

agtggtcgga tcttctcaac taccccagat gggcaaacag acgtcgtgag ctgcaatcta 420

agtacccgct tctgcttcgc tccactctgc tctctgccat gcgagctggt cctgttctat 480

atgttgagac gtggccgaat atgatttctg gacgattagc tgattggttt atgtcccaat 540

atggcaataa tttcgttgac atgtgtgcta ggttgaccca gtcttgttcg aacatgcctg 600

ttgaacctga tgggaattat gatcaacaga tgcgtgcttt aattagtttg tggcttctgt 660

catacattgg ggtaatcaac caaaccaaca ccatcagcgg tttctacttc tcctcaaaga 720

ctcggggtca agcgttggac agttggactt tgttctatac cacgaatact aatcgtgtcc 780

aaattacgca gagacatttt gcttatgtgt gcgcccgatc tcctgattgg aacgtggaca 840

aatcatggat cgctgctgcg aacttaaccg ccattgttat ggcttgccgt caaccgccga 900

tgtttgctaa tcaaggcgtc attaatcagg cgcagaaccg acccggattc tccatgaatg 960

gggggacgcc cgtccacgag ctcaacttac ttactactgc gcaagagtgc atcaggcagt 1020

gggtggtagc aggcttggtg tcggcagcaa aggggcaagc actaacgcag gaagctaatg 1080

acttctcaaa cctcatccag gcggatctag gccagatcaa ggcgcaggac gacgctttgt 1140

acaatcagca gccgggatac gcgaggagaa taaaaccttt cgttaatggt gactggacac 1200

caggtatgac cgctcaagct ctggccgttc tagccacttt taccgcctag gcgtagggtc 1260

gtacgctgcc cgagtccagc cctccggcag cccgtggaga tcggacgagc gtcgtgtagg 1320

ga 1322

<210> 11

<211> 1202

<212> DNA

<213> S3 Gene fragment

<400> 11

gctttttgag tccttagcgt gcaagccgca atggaggtac gtgtgccaaa ctttcactcg 60

ttcgttgaag gaataacatc tagctacttg aagactcctg cttgctggaa tgcacaaaca 120

gcttgggata ctgtgacctt tcacgtccct gatgtaatta gagttggtaa cgcctactgt 180

tgttctcaat gttgtggtgt actctactac gggactctgc cctcggacgg taattatttc 240

cctcatcaca agtgtcatca gcaacagttt aggactgata ctccactgct tcgatacgtg 300

cgcattggta gaaccactga gcatctgatg gatcaatatg ctgtcgctct ggagtccatt 360

gctgaacact atgacgagat tagtcaacgt atggtcgatg agccagagaa tgacgaggtt 420

acacctcttg atatcgttac gcgtaccgaa tctatcagga gtgacaaggc agtcgaccca 480

gacttttgga catacccact tgagcggcgt tctgatgatt ctcgtagaga catcgcctca 540

gcatgctgga aaatgattga cgcgtcggcg cgtagtctca ctcttccaaa ttgcctcgtc 600

tccccctctt tgcactctcg ctccgtcttt ggtcagatgc aaacgaccac cactatatac 660

gatgttgcgg catcgggaaa ggccgttaag ttttcaccaa tggttgctac actagcgcaa 720

cgtgatgctg gccctgtaat gcttgcgaat gctgacccgg cggaaggcgt gtactctttc 780

tggacgtcgc acttcgcttt ctcaccgctc atcggcggag ttggaattac gggacagtac 840

gctcgtgagt cgtaccatca agtgggtcat ccagtgattg ggagtggtaa gaaggcatcg 900

cattacagga atctgttcat ggaagcgtgg cgcgggtggt cgaagtcagc tttcgcatgt 960

gctactggaa tggagccagc tgaatgtgaa tctcgtctga gaggacacgc tcgtactatg 1020

ctcggacgct ctctgccgcc cgtttgtgac gatgatgttg ctcagcagtc tggcgcggta 1080

ctgacttcgc tgcagaaaac gaacaagttc accgttgtgg agtgtggttg gtaagtacct 1140

ccgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cgcctattca 1200

tc 1202

<210> 12

<211> 1192

<212> DNA

<213> S4 Gene fragment

<400> 12

gctttttgag tccttgcgca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60

catccggcgc agctggtcag actgttttta gaaactttta cttactacga tgcaatatct 120

cagctgacgg tcgtaatgca acaaaagctg tgcaatccca ttttccattc ctttctcgtg 180

ctgtccgttg cctatctcct ctagccgctc attgtgctga taggactctt cgtcgtgaca 240

atgtgaaaca aattcttact cgtgagctgc catttccatc ggatttaatc aattacgcac 300

atcatgtgaa ctcatcctcc cttactactt ctcagggtgt cgaggcggca cgtctagtgg 360

cccaagtcta tggagaacag ctatcgtttg atcacattta tcccactggt tccgcaactt 420

actgccctgg agcgattgct aatgcgattt cccgtatcat ggctggtttt gtgccccacg 480

aaggtgacaa ctttaccccg gacggttcta ttgactatct cgccgccgac ctggtcgcgt 540

ataagttcgt gctcccttac atgctagata ttgtggacgg acgtccgcag attgttcttc 600

catcacacac tgttgaggag atgctgtcca acacgagttt gcttaattcg attgacgctt 660

catttggtat tgaatcgaag agcgatcaac gcatgacccg tgacgcggct gaaatgagtt 720

ctcgctcact taatgagctt gaggatcatg agcagagggg tcgaatgcct tggaaaatca 780

tgacggcaat gttcgcggcg caattgaagg tggagttgga cgccctagct gatgagcgcg 840

ttgaatctca ggctaacgct catgtgacat cttttgggtc tcgtctgttc aaccaaatgt 900

ctgcttttgt cccaattgat cgtgagttga tggagctggc tctactcatc aaagagcagg 960

gtttcgcaat gaatccaggg caagtcgcat ctaaatggtc gctgatacga cgatctggcc 1020

ccactcgccc gctatcaggc gcacgccttg agatcaggaa tggcaactgg acaattcgtg 1080

aaggtgacca gacgcttctg tctgtctccc cagctaggat ggcgtaaacg ggacccatgg 1140

tgcgggtgag gggccgccac accctctgcc gcgacctgga ctcttattca tc 1192

Claims

1. A strain of duck reovirus has a preservation number of: CCTCC NO: v201818.

2. Use of the duck reovirus of claim 1 in the manufacture of a vaccine for the prevention of duck reovirus disease; the duck reovirus disease is caused by a duck reovirus disease with a preservation number of CCTCC NO: v201818 by duck reovirus infection.

3. The use according to claim 2, wherein the vaccine is an inactivated vaccine or an attenuated live vaccine.

4. The use according to claim 3, wherein the vaccine is an inactivated vaccine.