KR20090039230A - Vaccine strain of duck hepatitis recently isolated in korea - Google Patents

Abstract

A new vaccine to duck hepatitis virus is provided to have excellence in resistance against epidemic duck hepatitis and lessen damage caused by the duck hepatitis. A method for producing vaccine(deposition No. KCCM10883P) of duck hepatitis virus having sequence of sequence No. 1 [SEQ ID NO:1] comprises: a step of diluting recently epidemic duck hepatitis virus in phosphate buffer; a step of inoculating 7-9 days old chicken embryo; a step of culturing the inoculated chicken embryo at 37°C for three days; and a step of collecting the cultured chicken embryo and performing subculture sequencially to adapt the chicken embryo. A process for manufacturing a new vaccine is performed with: the step of attenuating the duck hepatitis virus; a step of testing restoration of attenuated virus; a step of searching defense ability of vaccine to selected duck hepatitis virus; a step of searching defense ability in each pathway; a step of searching minimum amount of the vaccine; and a step of safety and amount of the vaccine.

Description

New strain of vaccine against duck hepatitis virus {Vaccine strain of duck hepatitis recently isolated in Korea}

The present invention relates to a new duck hepatitis virus vaccine, more specifically, recently inoculated in phosphate buffered duck hepatitis virus in Korea inoculated in 7-9 days old chickens and incubated for 3 days at 37 ℃ after The present invention relates to a duck hepatitis virus vaccine strain prepared by continually acclimating to chick embryos, collected and emulsified with phosphate buffer. The novel duck hepatitis virus vaccine according to the present invention was capable of exerting 100% protection against duck hepatitis, which is not recently observed in Korea when inoculated in 1 day old young ducks.

Duck hepatitis is a very deadly and rapidly spreading viral disease in young ducks caused by three different viruses called duck hepatitis viruses type 1, 2 and 3. Duck hepatitis virus type 1 is an acute disease of liver bleeding with hemorrhagic spots. Since it was first reported in the United States in 1945, it has been around the world and has also occurred in nearby China, Taiwan, and Japan. Duck hepatitis virus type 2 first developed in the United Kingdom in 1965, disappeared in 1969 and then again in 1983-1984, and has not been reported since 1985, but only in the United Kingdom. Hepatitis virus type 3 was reported in 1969 in the United States and the disease was reported in the United States.

Duck hepatitis was first reported in Chonnam in Korea in 1985 and has been causing a lot of damage to duck farms. Duck hepatitis, which was prevalent in Korea until 2002, was mainly caused by duck hepatitis virus type 1, and a hepatitis vaccine for this type was developed and marketed since 2001. Hepatitis cannot be prevented.

Table 1 below shows cases of duck hepatitis diagnosed in duck mortality commissioned by the Veterinary Medical Quarantine Service, and shows the case of damage caused by duck hepatitis virus in farms vaccinated against duck hepatitis since 2001. have.

¹⁾ () is the lane number of the multiplex PCR picture shown in FIG.

²⁾ (vaccine) is a case of duck hepatitis on a farm that has been vaccinated.

* The blue and black numbers are the preservation of the current virus and also the differential diagnosis of the viruses separated by multiple PCR using the virus.

Black and blue in Table 1 are samples used for multiple PCR of FIG. As can be seen in Table 1, damage to duck hepatitis occurred even in the farm inoculated with the existing vaccine. In order to find the cause, we analyzed the genes related to the antigenicity of the viruses isolated in Korea and the recently isolated duck hepatitis virus, which are currently used as vaccine strains. It was found that virus type 1 and genotype were different (see FIGS. 1A and 1B), and antiserum for each virus was produced and cross-serum neutralization test with each virus was applied to the embryo. The serotype neutralization ability between the two viruses was 220 to 550 times, indicating that the serotypes between the two viruses were also different (Table 2).

Cross-serum neutralization test of duck hepatitis virus type 1 and recent isolated domestic hepatitis virus Antiserum virus AP-04203P60 DRL-62P20 AP-04114 > 1 0.0025 ^* AP-04203 One 0.0045 DRL-62 0.0028 ^a One DHV-HS 0.0018 > 1

* r  value: heterologous serum Chinese titan /the same kind Serum neutralizing titer

In the cross-serial neutralization test of duck hepatitis virus type 1 (DRL-62 and DHV-HS) and domestically isolated duck hepatitis virus (AP-04114 and AP-04203), antisera of 2 weeks of domestic isolated duck hepatitis virus was used for SPF chicken. In addition, antiserum against the currently marketed parental hepatitis vaccine parent virus (DHV-HS) and duck hepatitis virus type 1 was prepared, and recently, the domestic isolated duck hepatitis virus and duck hepatitis virus type 1 standard type were prepared. The results of cross-serial neutralization experiments in the fetuses (Table 2) showed that the serotypes were different between the two viruses.

As a result of the analysis, it was confirmed that the duck hepatitis virus, which is currently popular in Korea, is a genotype and serologically different virus from the duck hepatitis virus type 1 which has been popular in the past. As a result, the genetically differential diagnosis of the recently developed two viruses has been applied to domestically developed duck hepatitis. As a result, hepatitis caused by the domestic epidemic duck hepatitis virus has been developed since 2001, and recently from 2003 to late June 2007 A total of 47 cases of suspected duck hepatitis were diagnosed with duck hepatitis at the Veterinary Medical Quarantine Service, and one of them (duck hepatitis caused by duck hepatitis virus type 1) has recently become popular in Korea. It was confirmed that the type of (Fig. 2). Therefore, there is an urgent need for the development of an effective new vaccine strain against a novel duck hepatitis virus, which is currently popular in Korea.

Therefore, the present inventors have revealed that the hepatitis virus, which is currently popular in Korea, is different from the existing type 1 virus and genotype and serologically, and developed such a new hepatitis virus vaccine strain through chick fetus to complete the present invention.

Accordingly, it is an object of the present invention to provide a new vaccine against the duck hepatitis virus, which is currently popular in Korea.

In the present invention, the latest hepatitis virus virus in Korea was diluted in phosphate buffer solution, inoculated in 7-9 days old chicks, incubated at 37 ° C. for 3 days, and the chicks were harvested and emulsified with phosphate buffer solution. Provided is a duck hepatitis virus vaccine strain prepared by passage.

In the present invention, a novel duck hepatitis virus vaccine strain resistant to the recent epidemic duck hepatitis lost pathogenicity to one-day-old duck by passage of the duck hepatitis virus, which has been popular in Korea in recent years, was inoculated with the latest epidemic It can be used to reduce the damage caused by the duck hepatitis virus.

In order to achieve the above object, the present invention in order to prepare a new vaccine against the hepatitis virus virus that is popular in Korea recently

1) attenuating the domestic isolated duck hepatitis virus;

2) testing the pathogenic return of the attenuated virus;

3) examining the protective ability of the selected duck hepatitis virus vaccine strain;

4) examining the defense ability of each vaccination route;

5) examining the minimum antigen content of the vaccine strain; And

6) examining the safety and weight of the vaccine strain;

Through the present invention was completed.

In the present invention, a new duck hepatitis virus vaccine strain prepared through the above procedure was deposited with the Korea Microorganism Conservation Center on October 10 to receive the accession number KCCM10883P.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self-evident.

Example 1

1) Attenuation of isolated duck hepatitis virus in Korea

Recently, hepatitis virus, which is popular in Korea, was inoculated into 7-9 days old chickens in the same manner as the existing literatures, incubated at 37 ° C for 3 days, and the chickens were harvested with 10% (w / v) emulsion. Passage was again.

First, the duck hepatitis virus AP-04203 ( GenBank accession number: DQ256134 ) was inoculated into a 12-day-old duck fetus, and the inoculated duck fetus was incubated in a 37 ° C. incubator. Inoculation was performed to determine the titer of the virus. As a result of investigating the pathogenicity of the virus, as shown in Table 3, the high pathogenicity of Pekin duck (80% in one-day-old duck, 60% in one-week-old duck and 33% in two-week-old duck) was high. It was a virus.

Investigation of pathogenicity of domestic isolates (AP-04203) before passage Inoculation date AP-04203 ¹⁾ Dead shooter % Mortality 1 day old 12/15 ²⁾ 80 1 week old 9/15 60 2 weeks old 5/15 33

¹⁾ Inoculation of 10 ^4.5 ELD ₅₀ per horse

* The titer of the virus was measured in 9 day old duck embryos.

^{2) Number of} deaths / inoculations

In Table 3, as the pathogenicity test of AP-04203, a domestic duck hepatitis virus isolate before passage, pathogenicity decreased as the age of duck increased.

The virus was serially passaged up to 120 eggs in eggs, and the titer of each passaged virus was measured using a 7-day-old fetus, and the pathogenicity of each passaged virus 10 ^3.5 ELD ₅₀ was compared. The results are shown in Table 4 below.

Pathogenicity Investigation of 1-Day-Ordered Ducks Experimental group (passage number) Dead shooter % Mortality    AP-04203 1 unit 16/20 80 10 9/20 45 40 spaces 2/20 10 50 spaces 2/20 10 60 spaces 0/20 0 80 spaces 0/20 0 90 spaces 0/20 0 100 spaces 0/20 0 120 spaces 0/20 0

¹⁾ Inoculated with 10 ^3.5 ELD ₅₀ per horse in 1 day old ducks

* The titer of the virus was measured through 7 day old fetuses

In the results of Table 4, the pathogenicity test in 1 day old ducks of chick embryos did not show pathogenicity in 1 day old ducks after the 60th passage.

As a result of intramuscular inoculation of 1-year-old Pekin duck with each passaged virus in eggs, mortality was 80% for the first passaged virus, 45% for the 10th passaged virus, and 10% for the 40th and 50th passaged virus. No pathogenicity was observed in 1-day-old ducks after the 60th passage. Genetic changes for each passaged virus are shown in Table 5 and we observed that pathogenicity was lost after 9 amino acid changes in the 3 nucleotide and polyprotein regions in the 5 'and 3' untranslated regions. Could. As a result, the virus passed over 60 years old was considered to be valuable as a new vaccine strain.

Investigation of genetic changes in chick embryo Passage number Gene region Pess rate (%) ^One) 5 ' UTR (nt) Polyprotein (aa) 3 ' UTR (nt) 1 unit - - - 80 (16/20) 10 - - - 45 (9/20) 30 spaces - 1920 (R → C) - NT 40 spaces - 164 (F → S), 1445 (Q → H) 7475 (G → A) 10 (2/20) 50 spaces - 479 (Q → H) Addition (A) between 7548 and 7549 10 (2/20) 60 spaces 407 (C → T) 353 (Q → H), 678 (R → W), 1200 (T → I), 1499 (L → S), 2234 (I → T) - 0 (0/20) 80 spaces - 1492 (K → N) - 0 (0/20) 90 spaces - 424 (A → S), 869 (K → N) 7718 (C → A) 0 (0/20) 100 spaces 117 (A → C) 860 (D → V), 1137 (S → F) - 0 (0/20) 120 spaces - 860 (D → V), 1137 (S → F), 359 (M → L), 1792 (I → V), 1827 (E → G) - 0 (0/20) change 2 nt 17 aa 3 nt -

Table 5 shows the genetic changes according to the pathogenicity of chick embryo acclimation.

2) Pathogenicity return test of attenuated virus

Inoculated with 10 ^5.0 ELD ₅₀ virus in 1-day-old Pekin duck by intramuscularly inoculated with the virus after passage in the 60's, which showed no pathogenicity. To prepare a 10% emulsion solution, the supernatant was continuously inoculated with the muscle supernatant 0.2ml supernatant in 1 day old Pekin duck (Pekin duck) was carried out a pathogenic return test, the results are shown in Table 6 below.

Pathogenic reversion test of passaged virus Passage Count Passage of 1 day old duck Passage 1 ¹⁾ Passage 2 ²⁾ Passage 3 ²⁾ Passage 4 ²⁾ Passage 5 ²⁾ Passage 6 ²⁾ P60 0/12 ³⁾ 0/12 0/12 6/12 NT ^{4 )} P80 0/12 0/12 0/12 0/12 3/12 5/12 P90 0/12 0/12 0/12 0/12 0/12 0/12 P100 0/12 0/12 0/12 0/12 0/12 0/12 P120 0/12 0/12 0/12 0/12 0/12 NT

¹⁾ Each passaged virus was intramuscularly inoculated into 1 day old ducks with 10 ^5.0 ELD ₅₀ for 5 days

²⁾ Inoculated with 0.2ml of 10% emulsion supernatant of liver and cecum tonsils of surviving duck in 1-day-old duck pathogenic return test for 5 days

³⁾ Killed / inoculated

⁴⁾ NT: not tested

In the results of Table 6, pathogenicity was first observed in the fourth day-old duck passage in the 60's passage, and virus passaged in the 80th passage was observed in the fifth-day duck passage. However, it was confirmed that pathogenicity did not return to the sixth one-day-old duck passage in the later passaged viruses from the 90s.

3) Investigation of defensive capacity of selected domestic isolate vaccines

Each virus passaged on a 1-day-old Pekin duck was inoculated with 10 ^3.0 ELD ₅₀ per horse using post 90 passages (90, 100, 120 passages) viruses that did not return pathogenicity. After 1 day of age, ducks were challenged with a recent domestic isolate (AP-04203P1) having pathogenicity, and their defense ability was examined, and the results are shown in Table 7 below.

Investigation on Defensive Capacity of Recently Selected Korean Vaccines Total number of chicks ^{1 )} AP-04203P1 ^{2 )} Dead shooter % Mortality 90's Passage 0/20 ³⁾ 0 100 generations 0/20 0 120 generations 0/20 0 Control 13/20 65

¹⁾ Inoculated each passaged virus with 10 ^3.0 ELD ₅₀ per horse.

²⁾ When the muscle inoculated with the 1 day old ducks 10 for AP-04203P1 virus after 3 days ^4.0 ELD ₅₀ also inoculated muscles.

³⁾ Killed / inoculated.

In the results of Table 7, it was confirmed that there is a protective effect against the recent duck hepatitis virus in all of the 90, 100 and 120 passages of the selected recent domestic isolate vaccine strains.

Therefore, the present invention was analyzed the nucleotide sequence of 100 passaged vaccine strains with excellent protection against the duck hepatitis virus of the new duck hepatitis virus vaccine strain (SEQ ID NO: 1, Figure 4a to 4d), which was October in Korea Microbial Conservation Center Deposited on 10th day was given accession number KCCM10883P.

4) Investigation of defense capacity by vaccination route

In the case of duck hepatitis, which occurs recently in Korea, duck hepatitis occurs mainly in young ducks of 3-7 days of age. In order to determine the protective ability according to the inoculation route of the Accession No. KCCM10883P vaccine strain according to the present invention, one day old duck was inoculated once with the vaccine strain 10 ^3.0 ELD _{50 according} to the present invention using the muscle inoculation method and the negative oral vaccination method. The results of the investigation are shown in Table 8 below.

Investigation of Vaccination Capacity of Vaccine by Passed Virus Vaccination Path Day after vaccination One 2 3 4 5 6 7 Dead Ducks (%) Inoculation 7/15 (47) 0/15 (0) 0/15 (0) 0/15 (0) 0/15 (0) 0/10 (0) 0/10 (0) Negative oral vaccination 14/15 (93) 12/15 (80) 3/15 (20) 3/15 (20) 0/15 (0) 0/10 (0) 0/10 (0) Control 15/15 (100) 14/15 (93) 13/15 (87) 11/15 (73) 11/15 (73) 6/10 (60) 7/10 (70)

1) Inoculate 10 ^3.0 ELD ₅₀ per day in ducks

2) AP-04203P1 virus attacks inoculated with 10 ^5.0 ELD ₅₀ per muscle

3) Number of dead deaths / inoculations

In the results of Table 8, the vaccine against the oral and intramuscular inoculation of the vaccine according to the present invention was examined for protection against pathogenic viruses in both inoculation pathways. However, when inoculated intramuscularly, 100% protection was observed after 2 days of vaccination, and in the case of oral vaccination with a negative dose, 100% protection was observed after 5 days of inoculation. I could confirm it. In other words, the route of inoculation of the new duck hepatitis vaccine was found to be effective in muscle vaccination in consideration of the damage to duck hepatitis in young ducks.

5) Investigation of minimum antigen content of vaccine strain

In order to examine the protective ability according to the vaccine strain content of Accession No. KCCM10883P according to the present invention, the vaccine was inoculated with muscle from 10 ¹ to 10 ³ ELD ₅₀ in 1 day old duck using 100 passaged virus (vaccine strain), and after inoculation. From day 1, the pathogenic virus was challenged with muscles and negatively. The results of the investigation of the defense ability are shown in Table 9 below.

Investigation of Protective Capacity According to Vaccine Inoculation Path ^{1 )} Vaccine content (ELD ₅₀ ) Attack ^{2 )} Pathway Vaccinations after vaccination 1 day later 2 days later 3 days later 4 days later Inoculation 10 ¹ muscle 13/15 (87) ³⁾ 11/15 (73) 6/15 (40) 6/15 (40) oral- 13/15 (87) 9/15 (60) 4/15 (27) 5/15 (33) 10 ² muscle 12/15 (80) 1/15 (7) 0/15 (0) 0/15 (0) oral- 13/15 (87) 2/15 (13) 1/15 (7) 0/15 (0) 10 ³ muscle 9/15 (60) 0/15 (0) 0/15 (0) 0/15 (0) oral- 8/15 (53) 0/15 (0) 0/15 (0) 0/15 (0) Control muscle 15/15 (100) 12/15 (80) 11/15 (73) 9/15 (60) oral- 14/15 (93) 13/15 (87) 10/15 (67) 8/15 (53)

¹⁾ Vaccination: 1 day old ducks vaccinated with the vaccine

²⁾ Inoculation: Inoculates the virus of AP-04203P1 with muscle and negative with 10 ^4.0 ELD ₅₀

³⁾ Number of Deaths / Inoculations (Fade Rate)

In the results of Table 9 above, the effect of the vaccine was not observed in the group inoculated with 10 ¹ ELD ₅₀ of the vaccine strain according to the present invention compared with the control group, but when the antigen content of the vaccine was 10 ² ELD _{50 or} more, vaccination 2 Since day, defense against pathogenic duck hepatitis virus has been observed.

6) Vaccine strain safety and weight gain investigation

Investigation of the minimum antigen content of Accession No. KCCM10883P vaccine strain according to the present invention showed that the minimum antigen content as a vaccine was 10 ^2.0 ELD ₅₀ . The minimum amount of 1000 dose of 10 ^5.0 ELD ₅₀ vaccine virus was used to investigate the safety and weight gain for 1 day old ducks, and the results are shown in FIG. 3. At this time, the safety was judged to be safe if death did not occur, and the weight gain was measured by the weight change value according to the age compared with the control group.

In the results of FIG. 3, no pathogenicity was observed in 1-day-old ducks, and the weight gain was examined on the 5th and 12th days after vaccination, similar to the control group. Therefore, the safety when inoculating 1 day old ducks with the vaccine of Accession No. KCCM10883P according to the present invention was confirmed.

[Test Example 1] Comparative experiment of the existing vaccine strain and vaccine strain according to the present invention

In order to determine the protective capacity of the vaccine and the existing vaccine strain of Accession No. KCCM10883P according to the present invention, 1 day old duck was inoculated with the existing vaccine line and Vaccine No. of the accession number KCCM10883P 10 ^3.0 ELD ₅₀ respectively from 2 days after the pathogenic virus (DHV-HS And AP-04203P1) and the mortality was measured after challenge inoculation, and the results are shown in Table 10 below. At this time, the existing vaccine strain used in this test was passaged once commercially available duck hepatitis vaccine (Green Cross Veterinary Medicine) was used after measuring the titer.

Vaccination vaccine ^a) Challenge v ⁾ DEF (survival / inoculation), ERA (%) 2DPI 3DPI Original vaccine DHV-HS 4/15, 26.7% 15/15, 100% AP-04203P1 10/15, 66.7% 12/15, 80% Vaccine strain of accession number KCCM10883P DHV-HS 2/15, 13.3% 3/15, 20% AP-04203P1 15/15, 100% 15/15, 100% Control DHV-HS 0/15, 0% 1/15, 6.7% AP-04203P1 4/15, 26.7% 7/15, 46.7%

^{a) The} vaccination content is 10 ^3.0 ELD ₅₀ immunized in 1 day old ducks.

^b) Inoculations inoculate 10 ^4.0 ELD ₅₀ into each pathogenic virus.

In the results of Table 10, 100 days of defense was observed from 2 days after inoculation of the vaccine number of the deposit number KCCM10883P according to the present invention, whereas 33.3% of deaths were observed on 2 days after inoculation with the existing vaccine. 20% mortality was also observed on day 3 after inoculation. Duck hepatitis is more sensitive and mortality occurs in younger ducks. Therefore, it is appropriate to inoculate one-day-old ducks to defend against duck hepatitis. Therefore, it was confirmed that the vaccine strain of Accession No. KCCM10883P according to the present invention is a new vaccine strain having a faster and better protective effect than the existing vaccine strain.

<References>

1. Crighton GW, Woolcock PR (1978) Active immunization of ducklings against duck virus hepatitis. Veterinary Records. 358-361.

2. Ding C, Zhang D (2007) Molecular analysis of duck hepatitis virus type 1. Virology. 361: 9-17.

Gough RE, Spackman D (1981) Studies with inactivated duck virus hepatitis vaccines in breeder ducks. 10: 471-479.

Hwang J, Dougherty E (1962) Serial passage of duck hepatitis virus in chicken embryos. Avian Diseases. 6: 435-440.

5.Kim MC, Kwon YK, Joh SJ, Lindberg AM, Kwon JH, Kim JH, Kim SJ (2006) Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae. J Gen Virol 87: 3307-3316.

6.Kim MC, Kwon YK, Joh SJ, Kim SJ, Tolf C, Kim JH, Sung HW, Lindberg AM, Kwon JH (2007) Recent Korean isolates of duck hepatitis virus reveal the presence of a new geno- and serotype when compared to duck hepatitis virus type 1 type strains. Archies of Virology. in press.

Sung HW, Kim JH (2000) Isolation of duck hepatitis virus and its attenuation in chicken embryos. Korean Journal of Veterinary Medicine. 40: 101-109.

8. Sung HW, Kim JH (2000). Development of a live vaccine strain of duck viral hepatitis using a Korean isolate. Korean Journal of Veterinary Medicine. 40: 110-116.

9.Tseng CH, Knowles NJ, Tsai HJ (2007) Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus. Virus Res. 123: 190-203.

10.Tseng CH, Tsai HJ (2007) Molecular characterization of a new serotype of duck hepatitis virus. Virus Res. 126: 19-31.

Woolcock PR (1991) Duck hepaitis virus type 1: studies with inactivated vaccines in breeder ducks. Avian Pathology. 20: 509-522.

1 is a phylogenetic tree of duck hepatitis virus by analyzing the genes of duck hepatitis virus prior to the present invention A) is a phylogenetic tree of duck hepatitis virus as a phylogenetic tree of duck hepatitis virus through the 3D gene sites well preserved in each Genus in the Picornaviridae family. Represents a new Genus. B) is a phylogenetic tree of the VP1 (structural protein) gene, which is a gene region related to the antigenicity of Picornavirus. The duck hepatitis virus, which has been popular in Korea recently, shows that the genotype of duck hepatitis virus is different.

Figure 2 is a view showing the differential diagnosis between duck hepatitis virus type 1 and recent domestic epidemic duck hepatitis virus through Multiplex PCR prior to the present invention. In the past, the virus isolated in Korea can identify two bands of 467bp amplification product common to duck hepatitis virus and 229bp amplification product of duck hepatitis virus type 1. However, the recent epidemic of the duck hepatitis virus in Korea can identify two bands of a common 467bp amplification product and a specific 311bp amplification product of the recent domestic epidemic duck hepatitis virus. In other words, Lane 1-5 is the standard type of duck hepatitis virus, domestic isolates and vaccines in the past, Lane 6-7 isolates in 2001, Lane 8 isolates in 2002, Lane 9-20 isolates from hepatitis virus in 2003-2006, Lanes 21-24 were isolated from the hepatitis virus isolates by the end of June 2007 (all 36 cases from 2003 to 2006 were identified with the latest pandemic duck hepatitis virus in all but one in 11 cases by the end of June 2007). Recently, the domestic hepatitis virus has been identified. Compared to Table 1, there is a recent outbreak of duck hepatitis on farms vaccinated on the market.

Figure 3 is a graph showing the results of examining the safety and weight gain of the novel duck hepatitis virus vaccine strain according to the present invention. Then when inoculated with the vaccine caution virus in the ducks one day old to the minimum vaccine content of 10 ^2.0 ELD ₅₀ 1000 times the concentration of 10 ^5.0 ELD ₅₀ of about a 100 passaged virus that measure changes in virulence and weight gain inoculated 5 days No difference was observed in weight gain with the non-vaccinated group at 12 days.

4A to 4D are nucleotide sequences of duck hepatitis virus vaccine strain of Accession No. KCCM10883P according to the present invention, and the wild type and other base portions (mutant portions) of the domestic duck hepatitis virus are darkly displayed.

<110> REPUBLIC OF KOREA (Management: Ministry of Agriculture and forestry, National Veterinary Research) <120> Vaccine strain of duck hepatitis recently isolated in Korea <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 7785 <212> DNA <213> duck hepatitis virus <400> 1 tttgaaagcg gctgtggtgt agaccatttt ctggcgcttg gttcagccag tatggtctca 60 ccgcagcctg cttggggtta ttcccaaacc ccttaattca acgcccagtc ctagcggtat 120 aggaccccct atccatttgt tttaccttta ccctccacta tatagtctgt tgcttggcta 180 ttgactttgg cttttgtttt tgacccacgt ctaagtctta atggattttg tagtggttag 240 cctaccaccc cttggccact aattcttggc tttctgtttg gggatccacc atatcttgga 300 ggtggtgctg aaatattgca agccacttgc tatgtgtgtg ttttccaaac atgaagcttt 360 ggtgcagtgg ttttggacaa ggaaaggcta gtgtttggtc tgggtataaa cccttgttgt 420 gaaacggatt accggtagta gcatctagtg gttccagtcc ataacatgag tgtatggtct 480 agagtggaca cagcttggat acagacacct ttagtattac tgggtgttcc agactagttc 540 ctgaggtacc aagttgtgag gggctatggg aaaacccctt tgatccacac tgcctgatag 600 ggtcgcggct ggtcgagtcc catacactat aataccagtt gactttcatg taatggacac 660 tctaactaaa aacattgaag atgaaaccgt caagattatt ggatcctgtg ccgagaaggc 720 acaagaagca atttctggtc ttggagctgt tgagagtgtt gcttctacta actctgtggt 780 tgccactgca aatgctacaa caacacaaac gattcctgat ccaacagatg gttccacaga 840 tgatttttat tcttgttcct atgaggttgg ggcccagggt gacaacatct cacgtttggt 900 ccatctacac accggacagt ggtctacaca gcatggagtt actacatgcc ttagatggtt 960 ggccactcct ggatgttttt atacagctaa cacccaacca gcatatggac aaactaggta 1020 ttttagattc atcaggtgcg gctaccactt ccgccttctt gtgaatgcac catctggtgc 1080 tgctggtggg ctaatgatgg tgtggatgcc ttatccatat tgccgggttc tcactggatc 1140 ttccaatgtg gatgcatcag tggatcgcag atcattgctg aatcttccct atgccatctt 1200 ggatctgcgc accaacactg aaattgacct ggttattcca tatgtaaact tcaggaatta 1260 tgttgaaatt aatgccacag aaagtgttgg tggggccata tgtgtctttg tgttgggagc 1320 ctttacacat gggtcaggaa cctccaatac tgttgactac actctttttg gcgagatgct 1380 tgaaactgac ttacaatgtc ctcggccctt caacgaccag ggtaagaaga aaccacggcg 1440 gaggccaatc cataaaccaa agagccctcc tcaggaatct cgcatcatta tccagcctgg 1500 accaggagct gcaaatctat ccaattccag tgtggtcacc atggctgaga gtgtggctct 1560 agctaatgaa ggtactgcag ttgactattc aacagctggg tgtgcatcat ctgtggatga 1620 tatagtcatg gtgctcagac gctggcagat tgtgggtgat tttctgtggg ctaacacagt 1680 gacccctggc aacaggatta gtaggtttca tgtggttttt aatcgaatgc caacttttgc 1740 tctctttttt gataagttcc aatattggag ggggtccctg gaggttaaat tattgacctt 1800 tggaagccag tttaatactg gccgctatca aatgtcatgg taccctgtct ctaatggaga 1860 acaaactctt gcccagtgtc agaattctgt gtttgttacc tgtgatgtct gtgctacgcc 1920 atccaccctt atcttgccct tcactaatac cacatggcga aaaagcacac gtgaaaatta 1980 tggctatata acctggcatg ttgtgaatcg cctgacagtc aactcaacat ctccatccac 2040 aattagctgt gtcattttga tgcgagttgg taaggatttt cagtttacag ctcccctgta 2100 tgggaacctg cagatggccg ccaatgatca gggtgattcc aatcagcttg gtgatgatga 2160 accagtgtgt tttctcaatt ttgagactgc aaatgtgcca atacaaggag agtcgcacac 2220 cttggtgaaa catttttttg gccgtcaatg gctggttcgt actgttcaac atactggtga 2280 ggtacaagag atggatttgc cagtgcctga ccggggtcat gcatctctgt tgcgcttctt 2340 tgcctatttc tctggagaag tgattctgac tattgtcaat aatggaacaa caccctgtat 2400 ggttgcacac tcttatacaa cagacaatct cacttctgaa tatgctgtta ctgccatggg 2460 gggtattctt atcccagcga actctgccaa gaatattagt atcccatttt attctgtcac 2520 acctctacgc cccactcgac ccatgccagc atctcagggg ggtggcttga cttttggcag 2580 gttgtatatc tggacacaat ctggaagcat ttctgttttt atgggcctcc acaagccagc 2640 tttgtttttt ccactgcccg catcaactta cacaacaccc acgcagtcga atgggcttga 2700 aattatgaat ctgcatgaac aatcagacca gccagactgc catctgtgta agatatgcag 2760 gaaaatgaag aaatggtctc gcaaccttcg cccatttcgc ttctgtttga gacttaaaac 2820 acttgccttt gagctccatt tggaaattga atcagaccaa tttcgaaatg ttagagacct 2880 cactactgag ggtgttgaac ccaatccagg ccctattatg gttgttggta aatcaggatc 2940 cggcaaaagt gtattgtgta acatattggc tgatgtaaat ctctttgagt ctaaattaac 3000 accctataca ctcacaacaa ctcaccagat tgaaactgtg actatctgtg gcaagcaagt 3060 gaccttaatt gatacaccag agataccaaa atatgatggc ccaatttctt gcttccttta 3120 tctcatcgaa gctggccgct ttaccaatga ggatgttatt ttcatgaaga caatgaggca 3180 atatttccct ggttttgaaa aatctactat tttagtcttg aacagggctg ttgaactccc 3240 caataatgat cagctcaacg attggattaa aactaatgga gaattggaat cacttgttcg 3300 tgcctgtgat ggccgggttg ccaaattcta tcgtggtaag attgccactg ccaagctgtt 3360 ggataaaatt gcagagttgc ctgagtatcg tgctcactta ccgaggttag tttacaaaga 3420 tagaaagatg taccgtcact atggtgtgca gtgtggtaat gttgtatttc acatggactc 3480 tgagaacata atgaaatctg cactcaatgg agaggtcacc atcaaacaag agaagtggaa 3540 tggcaattgg aaaccagcca gtgagcacat gcaaagcaca gcatcaatct atttaaaatc 3600 tgacacaatg ccaaaattta aattttctgt tgatgataat tgtgagactt gggcaagaca 3660 attgctgggt gattatggac ccacacaggg aacaatcttg aaagagcgcc tcacgtgggc 3720 tgcagcattg ggttttttta tgacaatgaa aatcacaact gaccaatcat ttcctgggaa 3780 agatgccata catactgttt tgactaaaat ttccaacttt atttttggtg gtcttgaaaa 3840 tgaagtggtg agaattgtca tccgcacagt gatacgcatt gtctgctatc taattttgta 3900 tatccattca ccaaatattg tcaccacagg gacgttggtt gcactgcttg ccctagatgc 3960 cactagtatg agtatggacc agggccttaa gactctctgt atgagtttgg ttgatgggga 4020 ttttggaaaa ttttgttcag tcttgttgca aaaaattcaa ttcgttgatg aggctgacct 4080 taaaaacacc attccctcat tcactgacat gatggaagac caatcaggca aacctactgg 4140 tcctaagact tttaatgatt ggaccacatg tgcaaaaaat gttcaatggt ggttagaatc 4200 ttttgtaaaa gttgtgaatt ggctaaaaga aaaagtgttt ccatctaaaa ctgaccccac 4260 cctacagtgg cttgaagatc atgaggagca tatttctatt atgttggcat tgtgtgatga 4320 acacttgtgc atgctccgga ctgataagga ctatatttgt gaacatacca ctcgtcctaa 4380 gcaccaaaaa ttggttgaga tggtctctgg caccttgaat caattaaatg gcatctccag 4440 tgctaaagat ttgtgcttga gattgcagca tgtcttaaat aagcttcatc aggtcaattt 4500 tgagccagaa ttggaatgga cacaccgacc cgaacctctt ggtatttgga tttcaggtgg 4560 gccaggtgtt gggaagagtt ttttatccaa ctatatagtt aaacaaatag caaagaaaag 4620 gcattggaag tcttatgcaa atccaacagg gtcaaagcat atggatgggt atgtttcaca 4680 ggaaatccat gtttttgatg attttggcca gaatcgtgaa gaggaggatt attctctcat 4740 ttgtaatttg atttctagtg ttccatttat aacaccaaaa gccagtgttg aagccaaggg 4800 cacccagtat cgtggcagac ttgtagtagt taccaccaac aggagagatt tcacctcatg 4860 caaactcacc gatcctgacg cactagagcg taggtttcca atccgactca atattaggcc 4920 ccttcagaaa tacaatcata aaggtaggtt ggatgttgct actgcaatga gggatggcag 4980 tctacataat ggcacttgct gggaacgtga cattggtggg ttgggccttg aacactggaa 5040 tccaatcaat ggagacacac ttgtagatga gatcctctca gagctgcatg ttagacatga 5100 agtggctaat tttatgaatc agggcaatgt gcgtaggcta agtgatttgg acacaatgtt 5160 tgaagaattg gatgaattaa aacttgattt tgattttgac cgcctggaag aacaggctag 5220 actctttgcc cgcccaaagg aaggtaaaat ttctaagttc agaacttggg tgaaagaatg 5280 tatcaataag attaaaggct tccttgagcg taacaaaact tggatacttg gaattggaac 5340 tcttggtacc attgtgtccc tcataacaat gtgtattcca ttggctcgta aatttactca 5400 atctatctat tcagcccaac caatggctaa gactctacca aaagacttca aagtggctgt 5460 gcagaagcat gtagaaaagc ttgaaagtgt gctccaggac cagagtggtc gtgtgaattt 5520 ccgccacata tgcaatagat tggtcaatgt ctctagtgaa aatgaagttg ccacaggatt 5580 ggcagttggt ggcaagtatg tcttaacttt tggacattct aagtttactc aattggactc 5640 catcagggat atggtcttta attcacctgc caaaggaaca ccaattactt atgatgggct 5700 accaacagat ctccaactct tggattgtga cataccccac caattcaaag atgtgtctaa 5760 gctcatagcc acagatgatt atcgaggtaa tggctggttg gtgtggaaag atgatgatca 5820 atatatgatt caagaggtca ctaaaatcag accctttggg caaactacaa ctgcttctgg 5880 cacaacatca tgccaaacat acatttataa ttgtaagaca ggcccaggtt catgtggtgg 5940 agtacttgta gcccttgttg gaggaaatct gaaaattctc gggatccaca ctagtggtaa 6000 tggcacaatg ggtgcaagta atcgcatctt ccctgtattc aatcaaggag ccattgttga 6060 gaaaaagtat tcaggaaaga tcctctacca ccagccccgc aagactgctt accaaaaatc 6120 accagtttat gaggattctc cttatgaacc tgctgttctt tctatcaatg accagcgcct 6180 tgcagtacca attgaggaca tggctaagaa agctagtgac aaatatattg gaaatacatt 6240 tgatccacca ccaccagctt ttcgattggc aaaaacacat gtagctgaaa aattggctaa 6300 agtgttgggt tctcatgatt gtgtatctta tgagcaggcc atttccagtg atgtaatacc 6360 aatgaattgg gacacctctc caggtattaa gtacaagggt gagacaaagt gccagctggt 6420 gctcaaatct tcttttaaac aggatgttat ggatcagatt cagtctccat ctacagtgtt 6480 tgtgtgttac ctcaaagatg agctacgtaa gaaggagaaa atcaaggaag gaaagactcg 6540 aggtattgag gcatgcaact ttgactacac agttgcgttc cgcatggtta tgggtgaaat 6600 ttattccaat atttatgatg acagctttat tttatcaggc tgtgctgttg gaattaaccc 6660 ttttgctgag tgggacaatc tgttagcgaa cctgcagcct tacaacttgt gtttggactt 6720 ttctggtttt gacgggtctc tgagtgctca aattttggaa gaagctgttg atgtgttgtc 6780 atttttccat aatgatccag cccttgtgaa gagaattcat gaaccaacca tttattcaac 6840 acactatgtc actgatgaga tttggaaggt ggaaggcggt atgtgctcag gatcaccatg 6900 tactactgtc ctaaattcaa ttgtgaacca gctggcatgt tacactgtgc ttgctgtgtt 6960 gggctatgac atcaatcagt gctatgttgt cagctatgga gatgactgtg ttttgtctgt 7020 gcctgagaag cgggatatta gtaaactatc ccattatttt aaacttttct ttggcatgac 7080 agccacagcc agcgacaagg ctagcgacat agcttggaga ggcccaatgg aaattgagtt 7140 tctgaaaaga acacctgcat ttctgcctga tactcgtaag atagttggag ttctggacaa 7200 ggaagttctt gaaggcaaga ttcagtggtg caaaggccca gaagcattca aacaacaatt 7260 ggattcattt ttcttagaag ctgccttgca tggtcctgaa tattacaact acatttgttc 7320 caagttgaaa gctcgctgtc ctgtcttgga aacacaacca tggggtgttg ccaagatgcg 7380 agcatatact gcctgcatga ttatataacc tgcaggcata gcagttccag ggatctgctg 7440 tagtcaatta aaacctgggc tcttcgtggt tacgaggttt tccacttctg tttcaaaaac 7500 ctccctctag ggcgtagtgt tgtgggatac cctagagtac acgtaaaaac acccaccggt 7560 ttggcaggat accgcagagc aactgcccag tgttggtcct actgtctgaa caggactact 7620 tttagaatag ttttgctaat caattccccg tctaggttga tttagttagt atttagtata 7680 agaccaatgg tgatataaag accacaccta atctccaatt tcggtgtgtg accctaagcc 7740 attggacttc tttactttac tattcctccc caccctaaaa aaaaa 7785  

Claims (1)

Duck hepatitis virus vaccine strain of Accession No. KCCM10883P comprising the nucleotide sequence of SEQ ID NO: 1.

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