KR101481986B1 - Live vaccine protecting against pathogenic avian influenza H5N1 and use thereof - Google Patents

Abstract

본 발명은 x-31 ca 바이러스의 내부 유전자와 조류인플루엔자 바이러스 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 또는 A/Vietnam/HN/31242/2007 유래의 HA 및 NA를 코딩하는 유전자를 6:2의 비율로 가지고 있는 재배열 바이러스에 관한 것이다. 본 발명에 따른 재배열 바이러스는 기존의 ca A/Ahn Arbor/6/60 바이러스를 모균주로 이용한 경우보다 훌륭한 면역원성을 가지며 1회의 투여만으로도 높은 항체가를 나타내는 효과가 있다. The present invention relates to an internal gene of x-31 ca virus and to a method for detecting an avian influenza virus A / Indonesia / 05/2005, A / Chicken / Korea / ES / 2003, A / Vietnam / 1203/2004 or A / Vietnam / HN / 31242/2007 Derived HA and NA at a ratio of 6: 2. The rearranged virus according to the present invention has a better immunogenicity than the case of using the existing ca A / Ahn Arbor / 6/60 virus as a parent strain and has a high antibody titer with only one administration.

Description

조류인플루엔자 예방 생백신과 이의 용도 {Live vaccine protecting against pathogenic avian influenza H5N1 and use thereof}Avian Influenza Prevention Living Vaccine and Its Use {Live vaccine protecting against pathogenic avian influenza H5N1 and use thereof}

본 발명은 x-31 ca 바이러스의 내부 유전자와 조류인플루엔자 바이러스 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 또는 A/Vietnam/HN/31242/2007 유래의 HA(hemagglutinin ) 및 NA(neuraminidase)를 코딩하는 유전자를 6:2의 비율로 가지고 있는 재배열 바이러스에 관한 것이다.
The present invention relates to an internal gene of x-31 ca virus and to a method for detecting an avian influenza virus A / Indonesia / 05/2005, A / Chicken / Korea / ES / 2003, A / Vietnam / 1203/2004 or A / Vietnam / HN / 31242/2007 It relates to a rearrangement with virus in the ratio of 2: the gene encoding the HA (hemagglutinin) and NA (neuraminidase) of the resulting 6.

최근 생백신의 안전성을 높이기 위한 방법으로서, 두 가지 이상의 약독화 방법을 한 가지 생백신에 동시에 적용하여 우수한 안전성을 지니도록 하는 방법이 제안되었다. 종래기술로서 바이러스에 저온적응(cold-adaptation) 형질을 도입함으로써, 인간의 체온인 36~37℃ 에서 증식을 억제시키는 방법이 있다 (Monto, A.S. et al., Evaluation of an attenuated, cold-recombinant influenza B virus vaccine. J Infect Dis 145, 57-64 (1982); Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006); Belshe, R.B. et al., Current status of live attenuated influenza virus vaccine in the US. Virus Research 103, 177-185 (2004). 다른 종래기술로는 독감바이러스의 단백질 중, 비구조단백질(nonstructural protein 1, NS1)을 제거하거나 변형시켜 바이러스의 독성을 감소시키는 방법이 있다. 또한, M2 이온채널 단백질의 제거, 헤마글루티닌(hemagglutinin, HA), 단백질 절단 부위의 변형 등의 방법을 통해 바이러스 독성을 약화시키는 방법이 개발되었으며, 최근에는 miRNA를 유전자사일런싱(gene silencing) 기작을 이용하여 세포내에서 바이러스의 유전자 복제효율을 감소시키는 방법이 개발되었다. Recently, as a method for enhancing the safety of a live vaccine, a method of simultaneously applying two or more attenuation methods to one live vaccine to provide excellent safety has been proposed. As a conventional technique, there is a method of inhibiting proliferation at a human body temperature of 36 to 37 DEG C by introducing a cold-adaptation trait into the virus (Monto, AS et al., Evaluation of an attenuated, cold-recombinant influenza B virus vaccine. J Infect Dis 145, 57-64 (1982); Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006); Belshe, RB et al., Current status of live attenuated influenza virus vaccine in the US. Virus Research 103, 177-185 (2004). Another conventional technique is to remove or modify the nonstructural protein 1 (NS1) in the protein of influenza virus to reduce the toxicity of the virus. In addition, methods for attenuating viral toxicity have been developed by methods such as removal of M2 ion channel protein, hemagglutinin (HA), modification of protein cleavage sites, etc. Recently, miRNAs have been used for gene silencing, Methods have been developed to reduce the gene replication efficiency of viruses in cells using a mechanism.

이에 본 발명자들은 저온 생장형 약독화 생백신 공여균주인 X-31 ca를 기반으로 6:2 형태의 조류인플루엔자에 대한 재배열 바이러스를 제조하였고, 다른 바이러스와의 재조합바이러스에도 예방효과를 부여할 수 있는 교차면역형 생백신에 관한 발명을 완성하게 되었다.
Therefore, the present inventors have prepared a rearranged virus for avian influenza strain 6: 2 on the basis of X-31 ca, a low-temperature growth attenuated live vaccine strain, and have been able to provide a preventive effect against recombinant viruses The inventors of the cross-immune type live vaccine were completed.

본 발명의 목적은 저온 적응된 약독화된 공여 독감 바이러스(x-31 ca 균주)의 6개의 내부 유전자와 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 또는 A/Vietnam/HN/31242/2007 유래의 HA(hemaglutinin) 및 NA(neuraminidase) 유전자를 포함하는 재배열 바이러스를 제공하는 데 있다.
It is an object of the present invention to provide a method for the identification of six internal genes of low temperature adapted attenuated donor flu virus (x-31 ca strain) and A / Indonesia / 05/2005, A / Chicken / / 2004 or A / Vietnam / HN / 31242/2007, which comprises the HA (hemaglutinin) and NA (neuraminidase) genes.

상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 6으로 표시되는 6개의 내부 유전자 및 서열번호 7로 표시되는 유전자와 상동성 95% 이상인 유전자를 포함하는 인플루엔자 재배열 바이러스를 제공한다. In order to achieve the above object, the present invention provides an influenza rearranged virus comprising six internal genes represented by SEQ ID NOS: 1 to 6 and a gene represented by SEQ ID NO: 7 and having a homology of 95% or more.

본 발명은 다른 구체예에서, 상기 재배열 바이러스에 서열번호 12로 표시되는 유전자와 상동성 95% 이상인 유전자를 추가로 포함하는 것을 특징으로 하는 재배열 바이러스를 제공한다. In another embodiment, the present invention provides a rearranged virus characterized by further comprising a gene having a homology of 95% or more with the gene represented by SEQ ID NO: 12 in the rearranged virus.

본 발명은 또한, 저온 적응된 약독화된 x-31 ca 균주의 서열번호 1 내지 6으로 표시되는 6개의 내부 유전자 및 서열번호 7로 표시되는 유전자와 상동성 95% 이상인 유전자를 재배열시켜서 인플루엔자 재배열 바이러스를 제조하는 방법을 제공한다.The present invention also relates to a method for regenerating influenza viruses by rearranging genes having homology of 95% or more with the six internal genes represented by SEQ ID NOS: 1 to 6 and the gene represented by SEQ ID NO: 7 of the attenuated x- Lt; / RTI > viruses.

본 발명은 또 다른 구체예에서, 상기 재배열 바이러스에 서열번호 12로 표시되는 유전자와 상동성 95% 이상인 유전자를 추가로 포함하는 것을 특징으로 하는 재배열 바이러스를 제조하는 방법을 제공한다. In another embodiment, the present invention provides a method for producing a rearranged virus, wherein the rearranged virus further comprises a gene having a homology of 95% or more with the gene represented by SEQ ID NO: 12.

본 발명에 있어서, 상기 인플루엔자 재배열 바이러스는 조류 인플루엔자 재배열 바이러스인 것을 특징으로 할 수 있고, 저온 적응된 형질을 나타내는 것을 특징으로 할 수 있으며, 약독화된 형질을 나타내는 것을 특징으로 할 수 있다. In the present invention, the influenza rearranged virus may be characterized as an avian influenza rearranged virus and may be characterized as having a cold-adapted trait, and may exhibit an attenuated trait.

본 발명에 있어서, 상기 서열번호 7로 표시되는 유전자와 상동성 95% 이상인 유전자는 서열번호 8, 서열번호 9, 서열번호 10 및 서열번호 11로 구성된 군에서 선택되는 것을 특징으로 할 수 있고, 상기 서열번호 12로 표시되는 유전자와 상동성 95% 이상인 유전자는 서열번호 13, 서열번호 14, 서열번호 15 및 서열번호 16으로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. In the present invention, the gene having a homology of 95% or more with the gene of SEQ ID NO: 7 may be selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, The gene having a homology of 95% or more with the gene of SEQ ID NO: 12 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

본 발명은 또 다른 구체예에서, 상기 재배열 바이러스를 포함하는 인플루엔자 백신 조성물을 제공한다.In another embodiment, the present invention provides an influenza vaccine composition comprising the rearranged virus.

본 발명에 있어서, 상기 백신은 조류 인플루엔자 바이러스 백신인 것을 특징으로 할 수 있고, 저온 적응된 형질을 나타내는 것을 특징으로 할 수 있으며, 또한 약독화된 형질을 나타낼 수 있다. 또한, 상기 백신은 이종 조류인플루엔자 바이러스에 교차 면역 반응을 형성하는 것을 특징으로 할 수 있으며, 백신은 경구 또는 비강내 투여용으로 제형화될 수 있다. In the present invention, the vaccine may be an avian influenza virus vaccine, may be characterized as having a cold-adapted trait, and may also exhibit an attenuated trait. In addition, the vaccine may be characterized in that it cross-reacts with a heterologous avian influenza virus, and the vaccine may be formulated for oral or intranasal administration.

본 발명에 있어서, 서열번호 1 내지 6은 x-31 ca 바이러스의 내부 유전자로 PB2(서열번호 1), PB1(서열번호 2), PA(서열번호 3), NP(서열번호 4), M(서열번호 5) 및 NS(서열번호 6)이고, 서열번호 8 내지 11은 각각 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 및 A/Vietnam/HN/31242/2007 유래의 HA 서열을 나타낸다. 또한, 서열번호 13 내지 16은 각각 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 및 A/Vietnam/HN/31242/2007 유래의 NA 서열을 나타낸다. 서열번호 7은 A/Indonesia/05/2005 유래의 HA 서열 중 일부이고, 서열번호 12는 A/Indonesia/05/2005 유래의 NA 서열 중 일부이다. 서열번호 8 내지 서열번호 11은 서열번호 7로 표시되는 유전자와 각각 100%, 97%, 96%, 95%의 상동성을 나타내고, 서열번호 13 내지 서열번호 16은 서열번호 12로 표시되는 유전자와 각각 100%, 97%, 97%, 95%의 상동성을 나타낸다.(SEQ ID NO: 1), PB1 (SEQ ID NO: 2), PA (SEQ ID NO: 3), NP (SEQ ID NO: 4), M (SEQ ID NO: 5) and NS (SEQ ID NO: 6), SEQ ID NO: 8 to 11 are A / Indonesia / 05/2005, A / Chicken / Korea / ES / 2003, A / Vietnam / HA sequence derived from HN / 31242/2007. SEQ ID NOS: 13 to 16 denote NA sequences derived from A / Indonesia / 05/2005, A / Chicken / Korea / ES / 2003, A / Vietnam / 1203/2004 and A / Vietnam / HN / 31242 / . SEQ ID NO: 7 is part of the HA sequence derived from A / Indonesia / 05/2005 and SEQ ID NO: 12 is part of the NA sequence derived from A / Indonesia / 05 / SEQ ID NO: 8 to SEQ ID NO: 11 show homology of 100%, 97%, 96%, and 95% with the gene of SEQ ID NO: 7, SEQ ID NO: 13 to SEQ ID NO: Homology of 100%, 97%, 97% and 95%, respectively.

본 발명에 있어서, "재배열"은 바이러스가 하나 보다 많은 모 바이러스 균주 또는 공급원으로부터 유래된 유전적 및/또는 폴리펩티드 성분을 포함하는 것을 의미한다. 예를 들어, 6:2 재배열이란 첫번째 모 바이러스로부터 유래된 6개의 바이러스 유전자 분절 및 두번째 모 바이러스로부터의, 예를 들어 헤마글루티닌 또는 뉴라미디다제를 암호화하는 하나의 상보성 바이러스 유전자 분절을 포함하는 것을 의미한다. In the present invention, "rearrangement" means that the virus comprises more than one parent virus strain or a genetic and / or polypeptide component derived from the source. For example, a 6: 2 rearrangement is defined as a sequence of six viral genes derived from the first parental virus and one complementary viral gene segment encoding from the second parental virus, e. G. Hemagglutinin or niramididase .

본 발명에 있어서, "저온 적응된" 및 "약독화된"의 의미는 당업계에 공지되어 있다. 예를 들어, "저온 적응된"이란 바이러스가 37℃에서의 그 성장의 100배 이내의 성장을 33 ℃에서 나타내는 것을 의미한다. 예를 들어, "약독화된"이란 바이러스가 마우스의 상기도에서는 복제하지만 폐 조직에서는 검출 불가능하며 동물에서 인플루엔자형 질병을 야기하지 않음을 의미한다. In the present invention, the meaning of "low temperature adapted" and "attenuated" is known in the art. For example, "low temperature adapted" means that the virus exhibits growth within < RTI ID = 0.0 > 100 C < / RTI > For example, "attenuated" means that the virus replicates in the mouse's upper tract but is not detectable in lung tissue and does not cause influenza-like illness in animals.

본 발명에 있어서, 헤마글루티네이션 저해 분석은 바이러스의 헤마글루티닌에 특이적인 항체를 항원인 헤마글루티닌과 반응시킴으로써 상기 항체에 의해서 바이러스와 적혈구의 결합에 의하여 형성되는 응집이 저해되는 정도를 측정하는 측정방법이다. In the present invention, the hemagglutination inhibition assay is a method in which an antibody specific to hemagglutinin of a virus is reacted with hemagglutinin, which is an antigen, to thereby inhibit the aggregation formed by the binding of virus with erythrocytes by the antibody Is measured.

본 발명에 있어서, 중화 항체는 바이러스에 결합하여 병원성을 저해하는 항체를 의미하고, 바이러스로 감염된 환자의 혈청 중에 바이러스의 증식을 억제하는 중화 항체의 정량을 진단에 이용할 수 있다.
In the present invention, a neutralizing antibody refers to an antibody that binds to a virus and inhibits pathogenicity, and a quantitative determination of a neutralizing antibody that inhibits the proliferation of virus in the serum of a patient infected with the virus can be used for diagnosis.

본 발명에 따른 x-31 ca 바이러스의 6개의 내부 유전자와 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 또는 A/Vietnam/HN/31242/2007 유래의 HA 및 NA 유전자를 포함하는 재배열 바이러스는 기존의 ca A/Ahn Arbor/6/60 바이러스를 모균주로 이용한 경우보다 훌륭한 면역원성을 가지며 1회의 투여만으로도 높은 항체가를 나타내는 효과가 있다.
A / Chicken / Korea / ES / 2003, A / Vietnam / 1203/2004 or A / Vietnam / HN / 31242/2007 The rearranged viruses containing the HA and NA genes have better immunogenicity than the case of using the existing ca / Ahn Arbor / 6/60 virus as a parent strain and have high antibody titers even with a single administration.

도 1은 MDCK 세포에서 H5N1 ca 백신의 저온 적응된 형질을 나타낸 그래프이다.
도 2는 마우스에서 H5N1 ca 백신(R-IN2005 및 R-CK2003)의 약독화된 형질을 나타낸 그래프이다.
도 3은 마우스에서 H5N1 ca 백신(R-VN2007)의 약독화된 형질을 나타낸 그래프이다.
도 4은 H5N1 ca 백신(R-IN2005 및 R-CK2003)의 면역원성을 나타낸 것이다.
도 5는 H5N1 ca 백신(R-VN2004)의 면역원성을 나타낸 것이다.
도 6은 이종 H5N1 바이러스에 대한 H5N1 ca 백신의 교차 면역원성을 나타낸 것이다.
도 7은 H5N2 바이러스의 치명적인 감염에 대한 보호 효과를 나타낸 것이다. Figure 1 is a graph showing cold adapted traits of H5N1 ca vaccine in MDCK cells.
Figure 2 is a graph depicting attenuated traits of H5N1 ca vaccine (R-IN2005 and R-CK2003) in mice.
Figure 3 is a graph depicting attenuated traits of the H5N1 ca vaccine (R-VN2007) in mice.
Figure 4 shows the immunogenicity of the H5N1 ca vaccine (R-IN2005 and R-CK2003).
Figure 5 shows the immunogenicity of the H5N1 ca vaccine (R-VN2004).
Figure 6 shows the cross-immunogenicity of the H5N1 ca vaccine against the heterologous H5N1 virus.
Figure 7 shows the protective effect of the H5N2 virus against a fatal infection.

이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

동물 세포의 준비Preparation of animal cells

293T 세포와 MDCK 세포는 10% FBS(Hyclone Laboratories)로 보충한 Hyclone Laboratories) 배지에서 배양하였다.
293T cells and MDCK cells were cultured in Hyclone Laboratories medium supplemented with 10% FBS (Hyclone Laboratories).

H5N1H5N1 백신의 제조 Manufacture of vaccines

재배열 H5N1 ca 백신을 제조하기 위하여 HA(hemaglutinin) 및 NA(neuraminidase)는 A/Indonesia/05/2005, A/Chicken/Korea/ES/2003, A/Vietnam/1203/2004 또는 A/Vietnam/HN/31242/2007 로부터 획득하였다. 각 HA 및 NA는 역유전학 기술을 이용하여 pHW2000 벡터에 클로닝하였다(Hoffmann E et al., A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 2000; 97:6108-13). 재배열 바이러스의 안정성을 위하여, HA 단편의 polybasic cleavage site(PQRESRRKKRG)를 monobasic cleavage site(PQREKRG)로 교체하였다.
(Hemaglutinin) and NA (neuraminidase) were used to prepare a rearranged H5N1 ca vaccine. A / Indonesia / 05/2005, A / Chicken / Korea / ES / 2003, A / Vietnam / 1203 / / 31242/2007. Each HA and NA was cloned into the pHW2000 vector using reverse genetics technology (Hoffmann E et al., A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 2000; 97: 6108-13) . For the stability of the rearranged virus, the polybasic cleavage site (PQRESRRKKRG) of the HA fragment was replaced with a monobasic cleavage site (PQREKRG).

H5N1H5N1 백신의 성장 동력( Vaccine growth power GrowthGrowth KineticsKinetics ))

시험관 내에서 H5N1 ca 백신의 저온적응된 형질을 분석하기 위하여, 37℃ 및 30℃의 두 가지 상이한 온도에서 성장 동력을 비교하였다. MDCK 세포를 0.01 감염다중도(MOI:multiplicity of infection)의 R-IN2005, R-CK2003, R-VN2004 또는 R-VN2007로 감염시킨 후, 37℃ 또는 30℃에서 배양하였다. 상등액을 매 24시간마다 수거하고, infectious viral titers는 33℃에서 MDCK 세포에서 플라크 어세이로 측정하였다. Viral titers는 세 번의 독립적인 실험으로부터 평균값으로 계산하였다.
To analyze the cold adapted traits of the H5N1 ca vaccine in vitro, growth powers were compared at two different temperatures of 37 ° C and 30 ° C. MDCK cells were infected with R-IN2005, R-CK2003, R-VN2004 or R-VN2007 with multiplicity of infection (MOI) of 0.01 and then cultured at 37 ° C or 30 ° C. Supernatants were collected every 24 h and infectious viral titers were measured by plaque assay on MDCK cells at 33 ° C. Viral titers were averaged from three independent experiments.

동물 실험Animal experiment

R-IN2005 및 R-CK2003 백신의 병원성은 10³, 10⁴, 또는 10⁵ PFU의 백신을 6주령 암컷 BALB/c mice에 비강으로 주입한 다음 측정하였다. 접종 후 2주 마다 mice로부터 혈청을 수거하여 헤마글루티네이션 저해 분석(hemagglutination inhibition (HI) assay), 중화 분석(neutralization (NT) assays)으로 항체 반응을 분석하였다. 기도와 뇌에서 H5N1 백신의 복제 정도를 측정하기 위하여 네 마리의 마우스를 10⁵ PFU의 각 백신으로 비강 접종하였고, 접종 후 1, 3, 5, 7일 후에 폐, 상기도(nasal wash) 및 뇌를 회수하였다. 백신에 의한 방어능은 A/Aquatic bird/Korea/W81/05 (ma81)(H5N2) 바이러스 5 MLD₅₀의 lethal challenge 후, 기도와 뇌에서 생존률과 viral clearance 값으로 측정하였다. 모든 실험 과정은 연세 실험동물 연구센터 동물실험윤리위원회(Institutional Animal Care and Use Committee (IACUC))의 가이드라인에 따라 수행하였다.
Pathogenicity of the R-IN2005 and R-CK2003 vaccines was determined by injecting 10 ³ , 10 ⁴ , or 10 ⁵ PFU of vaccine into the nasal cavity of 6-week-old female BALB / c mice. Serum was collected from the mice every two weeks after inoculation and the antibody response was analyzed by hemagglutination inhibition (HI) assay and neutralization (NT) assays. Four mice were inoculated intranasally with 10 ⁵ PFU of each vaccine to determine the degree of replication of the H5N1 vaccine in the airways and brain. After 1, 3, 5, and 7 days after inoculation, Respectively. The survival rate and viral clearance were measured in the airway and in the brain after lethal challenge of AML / Aquatic bird / Korea / W81 / 05 (ma81) (H5N2) virus 5 MLD ₅₀ . All experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).

바이러스 중화 분석(Virus neutralization analysis NeutralizationNeutralization ( ( NTNT ) ) AssayAssay ))

열-불활성화된 혈청을 PBS와 함께 96-웰 플레이트에서 2배 단계희석하였다. 인플루엔자 바이러스에 대한 중화 항체의 tites 분석을 위하여 인플루엔자 바이러스(100 PFU)와 희석한 혈청을 동일한 부피(50μl)로 첨가하고, 플레이트를 37℃에서 1 시간 동안 배양하였다. 그 다음, 플라크 분석을 위하여 이 혼합물을 융합성 MDCK 세포를 포함하고 있는 12-웰 플레이트에 흡착시켰다. 중화 항체 타이터(titer)는 대조군과 비교하여 50% 플라크 감소를 나타내는 희석률로부터 계산되었다.
Heat-inactivated sera were diluted 2-fold in 96-well plates with PBS. Influenza virus (100 PFU) and diluted serum were added in the same volume (50 [mu] l) for analysis of neutralizing antibodies against influenza virus, and plates were incubated at 37 [deg.] C for 1 hour. This mixture was then adsorbed onto a 12-well plate containing fusogenic MDCK cells for plaque analysis. Neutral antibody titer was calculated from the dilution rate showing 50% plaque reduction compared to the control.

헤마글루티네이션Hemagglutination 저해 분석 Inhibition analysis

혈청을 수용체 파괴효소(receptor destroying enzyme)으로 처리하고 56℃에서 1 시간 동안 열을 가하였다. 그 다음, 혈청을 96-웰 플레이트에서 PBS로 2배 단계 희석하였다. 인플루엔자 바이러스의 4개의 헤마글루티네이션 유닛을 희석된 혈청에 첨가하고, 플레이트를 37℃에서 1 시간 동안 배양하였다. 배양한 후 1%의 cRBC을 동일한 부피로 첨가하고 4?에서 1 시간 동안 배양하였다. HI 타이터는 헤마글루티네이션을 완전히 억제하는 가장 높은 희석률로부터 계산하였다.
Serum was treated with receptor destroying enzyme and heated at 56 ° C for 1 hour. Serum was then diluted 2-fold with PBS in 96-well plates. Four hemagglutination units of influenza virus were added to the diluted serum and the plates were incubated at 37 DEG C for 1 hour. After incubation, 1% cRBC was added in the same volume and incubated at 4? For 1 hour. The HI titer was calculated from the highest dilution rate that completely suppressed hemagglutination.

통계 분석Statistical analysis

통계적 중요성을 확인하기 위하여 두 가지 다른 그룹을 비교할 때 Student's t-test를 사용하였다. p < 0.05 값을 유의적으로 의미있는 것으로 하고, 모든 에러 바(error bar)는 표준 편차를 나타낸다.
When comparing two different groups to identify statistical significance Student's t-test was used. Values of p <0.05 are considered significant, and all error bars indicate standard deviation.

H5N1H5N1 caca 백신의 저온 적응 형질의 확인 Identification of low temperature adaptive traits of vaccines

x-31 ca 균주를 유전적 백본으로 하여 A/Indonesia/05/2005 및 A/Chicken/Korea/ES/2003 유래의 HA 및 NA 유전자를 포함하는 6:2 재배열 바이러스 R-IN2005 및 R-CK2003를 제조하고, 이들 바이러스가 저온 적응 형질을 유지하고 있는지 확인하기 위하여 이들의 저온에서의 성장 특성을 실험하였다. MDCK 세포를 0.01 MOI의 R-IN2005 및 R-CK2003로 감염시킨 후, 30℃와 37℃에서 배양하였다. 감염 후 24 시간마다 상등액을 수거하고, MDCK 세포에서 플라크 분석으로 viral titer를 계산하였다. Using the x-31 ca strain as a genetic backbone In this study, 6: 2 rearranged viruses R-IN2005 and R-CK2003 containing HA and NA genes derived from A / Indonesia / 05/2005 and A / Chicken / Korea / ES / 2003 were prepared, And their growth characteristics at low temperatures were tested to confirm that they were retained. MDCK cells were infected with 0.01 MOI of R-IN2005 and R-CK2003, and then cultured at 30 占 폚 and 37 占 폚. The supernatant was collected every 24 hours post infection and viral titer was calculated by plaque analysis in MDCK cells.

R-IN2005 감염 후 24시간 경과시, 37℃에서의 viral titer는 30℃에서 보다 100배 더 높은 수치를 나타내었으나 더 증가하지는 않았다(도 1의 A). 반면에, 30℃에서의 viral titer는 처음 48 시간 동안 꾸준히 증가하였고 48 시간 경과시에는 37℃에서의 viral titer 값보다 10배 더 높은 값을 나타내었다. R-CK2003의 경우, 30℃에서 37℃ 보다 약 10배 더 높은 viral titer 값을 나타내었고, 두 온도 간에 더 확연한 viral titer 값의 차이를 보였으며 감염 후 지속적으로 값의 차이가 나타났다. 결론적으로, MDCK 세포에서 R-IN2005가 R-CK2003 보다 100배 정도 우수하게 복제되는 것을 알 수 있고, 이러한 결과는 R-IN2005와 R-CK2003 두 가지 H5N1 ca 백신이 모체인 x-31 ca 균주로부터 저온적응 형질을 이어 받았음을 의미한다.
At 24 hours after infection with R-IN2005, the viral titer at 37 ° C was 100-fold higher than at 30 ° C, but not further (Figure 1 A). On the other hand, the viral titer at 30 ° C steadily increased for the first 48 hours, and it was 10 times higher than the viral titer at 37 ° C for 48 hours. In the case of R-CK2003, the viral titer value was about 10 times higher than that at 37 ° C at 30 ° C. The difference in viral titer between the two temperatures was constant. In conclusion, it can be seen that R-IN2005 is replicated 100 times better than R-CK2003 in MDCK cells, and this result shows that the two H5N1 ca vaccines of R-IN2005 and R-CK2003 Which means that they have inherited cold adaptation traits.

마우스에서 R-In mice, R- IN2005IN2005 및 R- And R- CK2003CK2003 의 병원성 확인Pathogenicity confirmation of

R-IN2005와 R-CK2003의 약독화된 형질을 동물 모델에서 확인하기 위하여, 한 그룹 당 여덟 마리의 마우스를 R-IN2005 또는 R-CK2003 10³, 10⁴, 10⁵ PFU, PBS로 비강 접종하였다. 10³ PFU의 R-IN2005로 접종한 경우, PBS 대조군과 비슷하게 접종 후 2일 후에 약간의 체중 감소가 나타났다(도 2의 A). 10⁴ 또는 10⁵ PFU의 R-IN2005로 접종한 마우스는 접종 후 3일째에 약 10% 정도 체중이 감소하였으나 4일째부터 꾸준히 회복하기 시작했다(도 2의 A). R-CK2003의 경우, 10⁵ PFU로 접종한 경우에는 약간의 체중이 감소하였으나, 모든 그룹의 마우스들은 접종 후에 생존하였다(도 2의 B). 고병원성 H5N1 인플루엔자 바이러스의 MLD₅₀값이 10 PFU 이하인 것을 고려하면, 재배열 바이러스에서는 와일드타입 고병원성 조류인플루엔자의 치사율이 사라지고 생체 내에서 적절히 약독화된 형질을 나타내는 것을 알 수 있다. 또한, R-VN2007의 약독화된 형질을 동물 모델에서 확인하기 위하여, 여덟 마리의 마우스를 R-VN2007 10³, 10⁴, 10⁵ PFU, PBS로 비강 접종하였다. 그 결과, 체중 감소가 관찰되지 않았고 이를 통해 R-VN2007의 약독화된 성질을 확인할 수 있었다(도 3).To confirm the attenuated traits of R-IN2005 and R-CK2003 in animal models, eight mice per group were nasally inoculated with R-IN2005 or R-CK2003 10 ³ , 10 ⁴ , 10 ⁵ PFU, PBS . When inoculated with 10 ³ PFU of R-IN2005, a slight weight loss was observed after 2 days of inoculation, similar to the PBS control (Fig. 2A). The mice inoculated with 10 ⁴ or 10 ⁵ PFU of R-IN2005 lost about 10% of their body weight on the third day after inoculation, but began to recover steadily from the fourth day (FIG. 2A). In the case of R-CK2003, slight weight loss was observed when 10 ⁵ PFU was inoculated, but all groups of mice survived after inoculation (FIG. 2B). Considering that the MLD ₅₀ value of the highly pathogenic H5N1 influenza virus is 10 PFU or less, it can be seen that the rearranged virus exhibits the attenuated mortality rate of the wild-type highly pathogenic avian influenza virus and the appropriately attenuated trait in vivo. In addition, eight mice were inoculated nasally with R-VN2007 10 ³ , 10 ⁴ , 10 ⁵ PFU, PBS to confirm the attenuated trait of R-VN2007 in animal models. As a result, weight loss was not observed and the attenuated nature of R-VN2007 was confirmed (FIG. 3).

감염 마우스의 폐, 상기도 및 뇌에서 R-IN2005 또는 R-CK2003의 복제 정도를 통해 약독화 정도를 확인하였다. 이전의 연구들은 몇몇 H5N1 바이러스가 비강을 통한 감염 이후에 마우스의 뇌에서 관찰되었다고 보고한 바 있다(Gao P et al., Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong. J Virol 1999; 73:3184-9). 4 마리의 마우스를 10⁵ PFU의 R-IN2005 또는 R-CK2003으로 비강 감염시킨 후, 접종 후 2일 마다 viral titration를 위하여 폐, 상기도 및 뇌를 마우스로부터 제거하였다. The degree of attenuation was confirmed by the degree of replication of R-IN2005 or R-CK2003 in the lungs, upper respiratory tract and brain of infected mice. Previous studies have reported that some H5N1 viruses have been observed in the mouse brain following infection via the nasal cavity (Gao P et al., Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong J Virol 1999; 73: 3184-9). Four mice were nasal infected with 10 ⁵ PFU of R-IN2005 or R-CK2003, and the lungs, kidneys and brain were removed from the mice for viral titration every two days after inoculation.

R-IN2005 및 R-CK2003 백신 모두에서 접종 후 3일째에 가장 높은 viral titer 값을 나타내며 폐에서 효과적으로 복제되었고, 그 이후에는 급속도로 값이 감소하는 것을 확인하였다(표 1). 반면에, 면역력을 가지고 있는 마우스의 상기도 또는 뇌에서는 복제가 전혀 측정되지 않았다. 이와 같은 H5N1 ca 백신의 복제 패턴은 그들의 공여 균주인 x-31 ca 균주에서 관측되는 것과 상당히 비슷함을 알 수 있었다(Lee K-H et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 2006; 24:1966-74).
R-IN2005 and R-CK2003 vaccines showed the highest viral titer on day 3 after inoculation, effectively replicating in the lungs, and then rapidly decreasing in value (Table 1). On the other hand, replication was not measured at all in the brain or brain of immunocompetent mice. The replication pattern of the H5N1 ca vaccine was found to be similar to that observed in the x-31 ca strain, which is a donor strain thereof (Lee KH et al., Characterization of live influenza vaccine strains derived from cold-adaptation of X -31 virus. Vaccine 2006; 24: 1966-74).

조직group 백신vaccine 평균 바이러스 타이터(Average Virus Titer ( MeanMean VirusVirus Titer) Titer)
±± StandardStandard DeviationDeviation ( ( SDSD ) () ( loglog ₁₀₁₀ PFUPFU // mlml ) ) onon 1일1 day 3일3 days 5일5 days 7일7 days 폐lungs R-IN2005R-IN2005 4.29±0.384.29 ± 0.38 5.06±0.115.06 ± 0.11 3.86±0.163.86 ± 0.16 1.74±0.31.74 ± 0.3 R-CK2003R-CK2003 2.33±0.272.33 ± 0.27 4.23±0.174.23 ± 0.17 3.29±0.163.29 ± 0.16 1.96±0.31.96 ± 0.3 상기도The above diagram R-IN2005R-IN2005 ND^a ND ^a NDND NDND NDND R-CK2003R-CK2003 NDND NDND NDND NDND 뇌brain R-IN2005R-IN2005 NDND NDND NDND NDND R-CK2003R-CK2003 NDND NDND NDND NDND

(ND: 관측되지 않음)
(ND: not observed)

H5N1H5N1 caca 백신의 면역원성 확인 Identification of vaccine immunogenicity

H5N1 ca 백신의 면역원성을 확인하기 위하여, PBS 및 10³, 10⁴, or 10⁵ PFU의 R-IN2005 또는 R-CK2003 백신으로 접종된 마우스로부터 접종 후 2주째 마다 혈청을 수거하고, 혈청 항체 반응을 HI 및 NT 분석으로 측정하였다. 혈청 HI titers는 측정시마다 접종 용량에 따라 증가하였고, 접종 후 8주가 되었을 때 가장 높은 수치를 나타내었다(도 4의 A). 이를 통해서 감염 조직에서 백신 바이러스가 빠르게 제거됨에서 불구하고 면역 반응은 오랜 시간 동안 지속됨을 알 수 있다. 마찬가지로 NT 항체 titer는 접종 후 4주째까지 급속도로 증가하여 8주가 되었을 때 가장 높은 수치를 나타내었다(도 4의 B). NT 항체 titer는 접종 용량에 따라 증가하지 않고 모든 용량에서 비슷한 정도로 유지되는 것을 알 수 있고, 이것은 모균주인 x-31 ca 균주의 NT titer 양상과 비슷하다(Lee K-H et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 2006; 24:1966-74). 이를 통해, R-IN2005 또는 R-CK2003의 가장 적은 용량의 접종만으로도 동종 균주에 대한 중화 활성을 촉진하기에 충분하고, 한 번의 접종으로도 우수한 항체 반응을 일으킨다는 것을 알 수 있다. 또한, R-VN2004의 면역원성을 확인하기 위하여, PBS 및 10³, 10⁴, or 10⁵ PFU의 R-VN2004 백신으로 접종된 마우스로부터 접종 후 2주째 마다 혈청을 수거하고, 혈청 항체 반응을 NT 분석으로 측정하였다. 그 결과, 혈청 NT titers는 시간 및 접종 용량 의존적으로 증가하였다. 다만, 그 값은 R-IN2005 및 R-CK2003 보다 훨씬 낮았다(도 5).
To confirm the immunogenicity of the H5N1 ca vaccine, PBS and &lt; RTI ID = 0.0 &gt; Serum was collected every two weeks after inoculation from mice inoculated with R-IN2005 or R-CK2003 vaccine of 10 ³ , 10 ⁴ , or 10 ⁵ PFU and the serum antibody response was measured by HI and NT assay. Serum HI titers increased with dose at each measurement and were highest at 8 weeks after inoculation (Fig. 4A). This shows that the immune response lasts for a long time, though the vaccine virus is rapidly removed from the infected tissue. Similarly, NT antibody titer increased rapidly up to 4 weeks after inoculation and reached the highest value at 8 weeks (Fig. 4B). NT antibody titer did not increase with dosing dose but remained similar at all doses and was similar to the NT titer pattern of the parent strain x-31 ca (Lee KH et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 2006; 24: 1966-74). Thus, even with the smallest dose of R-IN2005 or R-CK2003 alone, it is sufficient to promote neutralizing activity against the homologous strain, It can be seen that even a single inoculation produces an excellent antibody reaction. Further, in order to confirm the immunogenicity of R-VN2004, PBS and Serum was collected from mice inoculated with 10 ³ , 10 ⁴ , or 10 ⁵ PFU of R-VN2004 vaccine every 2 weeks after inoculation and the serum antibody response was measured by NT assay. As a result, serum NT titers increased in a time and dose-dependent manner. However, the values were much lower than those of R-IN2005 and R-CK2003 (Fig. 5).

H5N1H5N1 caca 백신의 교차 면역원성 확인 Identification of cross-immunogenicity of vaccine

R-IN2005(clade 2.1), R-CK2003(clade 2.5) 및 R-VN2004(clade 1) H5N1 ca 재배열 백신 균주을 이용하여 교차 NT 항체 titer 값을 측정하였다. 그 결과, R-IN2005에 의해 형성된 혈청 항체가 이종 R-CK2003(clade 2.5)을 중화하는 데 발견되었고, 동종 백신 균주에 대하여 NT titer를 유도하는 정도로 효과적으로 기능하는 것을 확인하였다. 한편, 항원과 관련하여 더 구별되는 R-VN2004(clade 1)에 대하여는 좀 더 낮은 정도의 교차 면역원성을 나타내었다(도 6의 A). 또한, R-CK2003(clade 2.5)의 접종에 따른 교차 면역원성을 확인한 결과, 동종 균주에 대한 면역원성 보다는 이종 균주에 대하여 약한 면역원성을 나타내었지만 접종 용량과 시간에 따라 증가하는 NT 항체 titer 값을 나타내었다(도 6의 B). Cross-NT antibody titers were measured using R-IN2005 (clade 2.1), R-CK2003 (clade 2.5) and R-VN2004 (clade 1) H5N1 ca rearranged vaccine strains. As a result, it was found that a serum antibody formed by R-IN2005 was found to neutralize heterologous R-CK2003 (clade 2.5) and effectively functioned to induce NT titer against the homologous vaccine strain. On the other hand, it showed a lower degree of cross-immunogenicity for R-VN2004 (clade 1), which is more distinctive in relation to the antigen (Fig. 6A). In addition, cross-immunogenicity of R-CK2003 (clade 2.5) was confirmed. As a result, NT immunoreactivity to heterologous strains was weaker than immunogenicity to homologous strains but NT antibody titer increased with dose and time (Fig. 6B).

이를 통해, H5N1 ca 백신은 이종 H5N1 균주에 대하여 교차 면역원성을 나타내고, 항원과 관련하여 더 가까운 균주에 대하여 상대적으로 높은 반응성을 나타냄을 확인할 수 있었다.
From these results, it was confirmed that the H5N1 ca vaccine exhibits cross-immunogenicity against the heterologous H5N1 strain and exhibits a relatively high reactivity to the more closely related strains with respect to the antigen.

이종 치명적인 항원투여(Heterologous lethal antigen administration ( LethalLethal ChallengeChallenge )에 대한 보호) Protection against

H5N1 ca 백신의 보호 능력을 확인하기 위하여, 마우스를 10³, 10⁴, 10⁵ PFU의 R-IN2005 또는 R-CK2003으로 접종하였다. 접종 후 4주 후에, 모든 그룹을 마우스 적응된 A/Aquatic bird/Korea/W81/05(ma81)(H5N2) 바이러스 5 MLD₅₀ 를 투여하였다(Song M-S et al., The polymerase acidic protein gene of influenza A virus contributes to pathogenicity in a mouse model. J Virol 2009; 83:12325-35). ma81 바이러스는 마우스의 폐에서 저병원성 조류 인플루엔자 서브타입을 분리해낸 마우스 적응된 악성 균주이다. 와일드타입 H5N1 바이러스의 인간 감염과 관련된 안정성 때문에 ma81 바이러스를 도전 균주로 대신 사용하였다. 백신을 접종하지 않은 마우스들은 H5N2 바이러스에 감염된지 7 내지 9일만에 모두 쓰러졌지만, 백신을 접종하여 면역력을 가진 마우스들은 10³ PFU로 접종한 경우에도 모두 살아 남았다(도 7). 따라서, H5N1 ca 백신 하나의 접종만으로도 이종 H5N2 감염에 대해 완벽한 보호능을 가지는 것을 알 수 있었다. To confirm the protective ability of the H5N1 ca vaccine, 10 ³ , 10 ⁴ , 10 ⁵ PFU of R-IN2005 or R-CK2003. Four weeks after inoculation, all groups were dosed with 5 MLD ₅₀ of the mouse adapted A / Aquatic bird / Korea / W81 / 05 (ma81) (H5N2) virus (Song MS et al., The polymerase acidic protein of influenza A virus contributes to pathogenicity in a mouse model. J Virol 2009; 83: 12325-35). The ma81 virus is a mouse-adapted malignant strain that has isolated the low-pathogenic avian influenza subtype from the lungs of mice. Because of the stability associated with human infection of the wild-type H5N1 virus, ma81 virus was used instead as a challenge strain. Mice that did not receive the vaccine fell all within 7 to 9 days of infection with the H5N2 virus, but all of the mice immunized with the vaccine survived even when inoculated with 10 ³ PFU (FIG. 7). Therefore, it was found that only one inoculation of H5N1 ca vaccine had complete protection against heterozygous H5N2 infection.

치명적인 감염에 대한 보호능에 대해 더 알아보기 위하여, 백신을 접종하여 면역력을 가진 마우스의 기도 및 뇌에서 감염된 바이러스의 복제 정도를 측정하였다. 10⁵ PFU의 R-IN2005 또는 R-CK2003 만으로 접종하고 4주가 지난 후, 모든 마우스들을 ma81 바이러스 5 MLD₅₀ 으로 감염시켰다. 접종 후 3일, 5일이 지났을 때, 마우스들을 희생시켜 폐, 상기도 및 뇌를 회수하였다. 감염된 바이러스는 면역력을 가진 마우스의 기도에서만 매우 제한적으로 복제된 것을 확인하였다. 면역력을 가지는 마우스의 폐에서 확인된 감염 바이러스의 titers는 백신이 접종되지 않은 마우스에서 보다 10^3-4 배 낮은 값이 나타났다(접종 후 3일). 더욱이, 면역력이 없는 마우스에서는 상기도에서 높은 viral titers를 나타낸 반면, 면역력을 가지는 마우스에서는 바이러스의 복제가 관찰되지 않았다(접종 후 3 내지 5일). 이러한 결과는 H5N1 ca 백신의 접종으로 면역력을 가지게 되면 기도에서 감염 바이러스를 빨리 제거한다는 것을 의미한다.
To better understand the protection against fatal infections, the vaccine was inoculated to measure the degree of replication of infected virus in the airways and brain of immunocompetent mice. After 4 weeks of inoculation with only 10 ⁵ PFU of R-IN2005 or R-CK2003, all mice were infected with ma81 virus 5 MLD ₅₀ . Three days and five days after the inoculation, the mice were sacrificed to recover the lung, upper respiratory tract and brain. The infected viruses were found to be replicated only very limitedly in the airways of immunocompetent mice. The titers of infectious viruses identified in the lungs of immunocompetent mice were 10 ^{3 to 4-} fold lower than in vaccinated mice (3 days post-vaccination). Furthermore, high viral titers were seen in the immunocompetent mice in the immunocompetent, whereas virus replication was not observed in immunocompetent mice (3 to 5 days after inoculation). These results indicate that immunization with the H5N1 ca vaccine quickly removes the infectious virus from the airways.

조직group 백신vaccine 평균 바이러스 타이터(Average Virus Titer ( MeanMean VirusVirus Titer) ±  Titer) ± SDSD ( ( loglog ₁₀₁₀ PFUPFU // mlml ) ) onon 3일3 days 5일5 days 폐lungs R-IN2005R-IN2005 2.8±0.14^a 2.8 ± 0.14 ^a ND^b ND ^b R-CK2003R-CK2003 2.05±0.16^a 2.05 ± 0.16 ^a NDND PBSPBS 6.02±0.356.02 ± 0.35 6.08±0.556.08 ± 0.55 상기도The above diagram R-IN2005R-IN2005 NDND NDND R-CK2003R-CK2003 NDND NDND PBSPBS 3.4±0.193.4 ± 0.19 3.7±0.343.7 ± 0.34 뇌brain R-IN2005R-IN2005 NDND NDND R-CK2003R-CK2003 NDND NDND PBSPBS NDND NDND

[비교실험예][Comparative Experimental Example]

x-31 ca 균주를 모균주로 사용한 본 발명에 따른 재배열 바이러스와 기존의 A/Ahn Arbor/6/60 를 모균주로 사용한 재배열 바이러스 간의 면역원성, 교차 면역원성을 비교하였다(Amorsolo L. Suguitan Jr. et al., Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets, September 2006, Vol.3). The immunogenicity and cross-immunogenicity of the rearranged virus according to the present invention using the x-31 ca strain as the parent strain and the rearranged virus using the existing A / Ahn Arbor / 6/60 as the parent strains were compared (Amorsolo L. et al. Suguitan Jr. et al., Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets, September 2006, Vol.

그 결과, 기존의 A/Ahn Arbor/6/60 를 모균주로 이용한 경우 보다 본 발명에 따른 재배열 바이러스가 우수한 면역원성과 교차 면역원성을 나타냄을 확인하였다(10⁶ TCID50 는 10^5.7-5.8 PFU에 상응하는 값을 의미한다). A/Ahn Arbor/6/60 를 모균주로 이용한 바이러스 백신을 10⁶ TCID50 접종한 경우, 동종 균주에 대하여 NT 타이터 값이 10-60인 반면 x-31 ca 를 모균주로 이용한 경우에는 그보다 약 10배 정도 증가한 480-620을 나타내었다. 또한, 이종 균주에 대하여도 A/Ahn Arbor/6/60 를 모균주로 이용한 경우 보다 x-31 ca 를 모균주로 이용한 바이러스 백신은 3-90배 높은 NT 타이터 값을 나타내었다.
As a result, it was confirmed that the rearranged virus according to the present invention exhibited excellent immunogenicity and cross-immunogenicity compared to the case of using the existing A / Ahn Arbor / 6/60 as a parent strain (10 ^{6 TCID 50} was 10 ^5.7-5.8 PFU Meaning a corresponding value). In the case of 10 ⁶ TCID50 vaccination using A / Ahn Arbor / 6/60 as the parent strain, the NT titer value of the homologous strain was 10-60, whereas when x-31 ca was used as the parent strain, Which is about 480-620. In addition, for the heterologous strains, antiviral vaccines using x-31 ca as parent strains showed NT titer values 3-90 times higher than those using parent strains A / Ahn Arbor / 6/60.

재배열 바이러스Rearranged virus 증가율
(fold increase)Growth rate
(fold increase) 백본 균주Backbone strain A/Ann Arbor/6/60 ca
(10⁶ TCID50)A / Ann Arbor / 6/60 ca
(10 &lt; ⁶ &gt; TCID50) X-31 ca
(10⁵ PFU)X-31 ca
(10 ⁵ PFU) Homologous strainHomologous strain NT titer: 10~60NT titer: 10 ~ 60 NT titer: 480-620NT titer: 480-620 NT titer: 8~10.3NT titer: 8-10.3 HI titer: 5~50HI titer: 5 to 50 HI titer: 72-80HI titer: 72-80 HI titer: 1.44~1.6HI titer: 1.44-1.6 Heterologous strain
(cross-reactivity)Heterologous strain
(cross-reactivity) NT titer: 10~14NT titer: 10 ~ 14 NT titer: 40~1,280NT titer: 40 ~ 1,280 NT titer: 2.8~91.4NT titer: 2.8 to 91.4

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Method for preparing cold adapted attenuated live vaccine of highly pathogenic avian influenza H5N1 <130> IPDB46147 <160> 16 <170> KopatentIn 2.0 <210> 1 <211> 2316 <212> DNA <213> x-31 ca virus_PB2 <400> 1 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg aaatctaatg 60 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc 120 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg 180 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat 240 gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta 300 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcagtat 360 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc 420 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat 480 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa 600 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg 660 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg 720 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg 780 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca 840 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga 900 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc 960 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag 1020 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggaaa tcttcaaaca 1080 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca 1140 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa 1200 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata 1260 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcagcgatt gaatcctatg 1320 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt 1380 gaacctatcg acaatgtgat gggaatgatt gggatattgc ccgacatgac tccaagcatc 1440 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg 1500 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa 1680 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta 1740 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccag tagaggccaa 1800 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg 1920 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat 2040 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg 2100 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat 2160 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg 2220 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280 aaaagaattc ggatggccat caattagtgt tgaata 2316 <210> 2 <211> 2341 <212> DNA <213> x-31 ca virus_PB1 <400> 2 agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60 ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat 120 gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag 180 ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca 240 ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt ggaggcgatg 300 gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgtattga aacgatggag 360 gttgttcagc aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact 420 ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt gttcagatca 480 aatggcctca cggccaatga gtctggaagg ctcatagact tccttaagga tgtaatggag 540 tcaatgaaca aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga 600 gacaatatga ctaagaaaat gataacacag agaacaatgg gtaaaaagaa gcagagattg 660 aacaaaagga gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag 720 agagggaagc taaaacggag aacaattgca accccaggga tgcaaataag ggggtttgta 780 tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca 840 gttggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat gatgaccaat 900 tctcaggaca ccgaactttc tttcaccatc actggagata acaccaaatg gaacgaaaat 960 cagaatcctc ggatgttttt ggccatgatc acatatatga ccagaaatca gcccgaatgg 1020 ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga 1080 aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg 1140 ctagcaagca tcgatttgaa atatttcaat gattcaacaa gaaagaagat tgaaaaaatc 1200 cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260 aatatgttga gcactgtatt aggcgtctcc atcctgaacc ttggacaaaa gagatacacc 1320 aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat 1380 gcacccaatc atgaagggat tcaagccgga gtcgacaggt tttatcgaac ctgtaagcta 1440 cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc 1500 acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct tcccagtttt 1560 ggggtgtctg ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac 1620 aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc 1680 aaagattaca ggtacacgta ccgatgccat agaggtgaca cacaaataca aacccgaaga 1740 tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc 1800 gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860 tgggaattga tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc 1920 agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca tggtccagcc 1980 aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatcga 2040 tccatcttga atacaagtca aagaggagta cttgaggatg aacaaatgta ccaaaggtgc 2100 tgcaatttat ttgaaaaatt cttccccagc agttcataca gaagaccagt cgggatatcc 2160 agtatggtgg aggctatggt ttctagagcc cgaattgatg cacggattga tttcgaatct 2220 ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag 2280 ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340 t 2341 <210> 3 <211> 2233 <212> DNA <213> x-31 ca virus_PA <400> 3 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca 120 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac 180 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg 240 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac 300 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac 360 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg 420 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg 480 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa 540 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt 600 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc 660 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat 720 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa 780 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat 840 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt 900 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga 960 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca 1020 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag 1140 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa 1200 tatgatagtg atgaaccaga attgaggtca cttgcaagtt ggattcagaa tgagtttaac 1260 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg 1320 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac 1380 tgcagagcca cagaatacat aatgaaggga gtgtacatca atactgcctt gcttaatgca 1440 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag 1500 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt 1680 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa 1740 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt 1800 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc 1920 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct 1980 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttgtcgt tcaggctctt 2040 agggacaacc tggaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta 2220 ccttgtttct act 2233 <210> 4 <211> 1565 <212> DNA <213> x-31 ca virus_NP <400> 4 agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtctcaaggc 60 accaaacgat cttacgaaca gatggagact gatggaggac gccagaatgc cactgaaatc 120 agagcatccg tcggaaaaat gattggtgga attggacgat tctacatcca aatgtgcacc 180 gaactcaaac tcagtgatta tgagggacgg ttgatccaaa acagcttaac aatagagaga 240 atggtgctct ctgcttttga cgaaaggaga aataaatacc ttgaagaaca tcccagtgcg 300 gggaaagatc ctaagaaaac tggaggacct atatacagga gagtaaacgg aaagtggatg 360 agagaactca tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat 420 ggtgacgatg caatggctgg tctgactcac atgatgatct ggcattccaa tttgaatgat 480 gcaacttatc agaggacaag agctcttgtt cgcaccggaa tggatcccag gatgtgctct 540 ctgatgcaag gttcaactct ccctaggagg tctggagccg caggtgctgc agtcaaagga 600 gttggaacaa tggtgatgga attggtcaga atgatcaaac gtgggatcaa tgatcggaac 660 ttctggaggg gtgagaatgg acgaaaaaca agaattgctt atgaaagaat gtgcaacatt 720 ctcaaaggga aatttcaaac tgctgcacaa aaagcaatga tggatcaagt gagagagagc 780 cggaacccag ggaatgctga gttcgaagat ctcacttttc tagcacggtc tgcactcata 840 ttgagagggt cggttgctca caagtcctgc ctgcctgcct gtgtgtatgg acctgccgta 900 gccagtgggt acgactttga aagggaggga tactctctag tcggaataga ccctttcaga 960 ctgcttcaaa acagccaagt gtacagccta atcagaccaa atgagaatcc agcacacaag 1020 agtcaactgg tgtggatggc atgccattct gccgcatttg aagatctaag agtattaagc 1080 ttcatcaaag ggacgaaggt gctcccaaga gggaagcttt ccactagagg agttcaaatt 1140 gcttccaatg aaaatatgga gactatggaa tcaagtacac ttgaactgag aagcaggtac 1200 tgggccataa ggaccagaag tggaggaaac accaatcaac agagggcatc tgcgggccaa 1260 atcagcatac aacctacgtt ctcagtacag agaaatctcc cttttgacag aaccaccatt 1320 atggcagcat tcaatgggaa tacagagggg agaacatctg acatgaggac cgaaatcata 1380 aggatgatgg aaagtgcaag accagaagat gtgtctttcc aggggcgggg agtcttcgag 1440 ctctcggacg aaaaggcagc gagcccgatc gtgccttcct ttgacatgag taatgaagga 1500 tcttatttct tcggagacaa tgcagaggag tacgacaatt aaagaaaaat acccttgttt 1560 ctact 1565 <210> 5 <211> 1027 <212> DNA <213> x-31 ca virus_M <400> 5 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact 60 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120 tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420 caacaggatg gggactgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact 540 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat 660 ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga 720 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780 gtgatcctct cactattgcc gcaaatatca ttgggatttt gcacttgaca ttgtggattc 840 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020 ttctact 1027 <210> 6 <211> 890 <212> DNA <213> x-31 ca virus_NS <400> 6 agcaaaagca gggtgacaaa aacataatgg atccaaacac tgtgtcaagc tttcaggtag 60 attgctttct ttggcatgtc cgcaaacgag ttgcagacca agaactaggt gatgccccat 120 tccttgatcg gcttcgccga gatcagaaat ccctaagagg aaggggcagt actctcggtc 180 tggacatcaa gacagccaca cgtgctggaa agcagatagt ggagcggatt ctgaaagaag 240 aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt tacctaactg 300 acatgactct tgaggaattg tcaagggact ggtccatgct catacccaag cagaaagtgg 360 caggccctct ttgtatcaga atggaccagg cgatcatgga taagaacatc atactgaaag 420 cgaacttcag tgtgattttt gaccggctgg agactctaat attgctaagg gctttcaccg 480 aagagggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactgctg 540 aggatgtcaa aaatgcagtt ggagtcctca tcggaggact tgaatggaat gataacacag 600 ttcgagtctc tgaaactcta cagagattcg cttggagaag cagtaatgag aatgggagac 660 ctccactcac tccaaaacag aaacgagaaa tggcgggaac aattaggtca gaagtttgaa 720 gaaataagat ggttgattga agaagtgaga cacaaactga agataacaga gaatagtttt 780 gagcaaataa catttatgca agccttacat ctattgcttg aagtggagca agagataaga 840 actttctcgt ttcagcttat ttagtactaa aaaacaccct tgtttctact 890 <210> 7 <211> 1659 <212> DNA <213> Influenza A virus (A/Indonesia/05/2005 (H5N1)) <400> 7 gatcagattt gcattggtta ccatgcaaac aattcaacag agcaggttga cacaatcatg 60 gaaaagaacg ttactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 120 ctctgcgatc tagatggagt gaagcctcta attttaagag attgtagtgt agctggatgg 180 ctcctcggga acccaatgtg tgacgaattc atcaatgtac cggaatggtc ttacatagtg 240 gagaaggcca atccaaccaa tgacctctgt tacccaggga gtttcaacga ctatgaagaa 300 ctgaaacacc tattgagcag aataaaccat tttgagaaaa ttcaaatcat ccccaaaagt 360 tcttggtccg atcatgaagc ctcatcagga gtgagctcag catgtccata cctgggaagt 420 ccctcctttt ttagaaatgt ggtatggctt atcaaaaaga acagtacata cccaacaata 480 aagaaaagct acaataatac caaccaagaa gatcttttgg tactgtgggg aattcaccat 540 cctaatgatg cggcagagca gacaaggcta tatcaaaacc caaccaccta tatttccatt 600 gggacatcaa cactaaacca gagattggta ccaaaaatag ctactagatc caaagtaaac 660 gggcaaagtg gaaggatgga gttcttctgg acaattttaa aacctaatga tgcaatcaac 720 ttcgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 780 gactcagcaa ttatgaaaag tgaattggaa tatggtaact gcaacaccaa gtgtcaaact 840 ccaatggggg cgataaactc tagtatgcca ttccacaaca tacaccctct caccatcggg 900 gaatgcccca aatatgtgaa atcaaacaga ttagtccttg caacagggct cagaaatagc 960 cctcaaagag agagcagaag aaaaaagaga ggactatttg gagctatagc aggttttata 1020 gagggaggat ggcagggaat ggtagatggt tggtatgggt accaccatag caatgagcag 1080 gggagtgggt acgctgcaga caaagaatcc actcaaaagg caatagatgg agtcaccaat 1140 aaggtcaact caatcattga caaaatgaac actcagtttg aggccgttgg aagggaattt 1200 aataacttag aaaggagaat agagaattta aacaagaaga tggaagacgg gtttctagat 1260 gtctggactt ataatgccga acttctggtt ctcatggaaa atgagagaac tctagacttt 1320 catgactcaa atgttaagaa cctctacgac aaggtccgac tacagcttag ggataatgca 1380 aaggagctgg gtaacggttg tttcgagttc tatcacaaat gtgataatga atgtatggaa 1440 agtataagaa acggaacgta caactatccg cagtattcag aagaagcaag attaaaaaga 1500 gaggaaataa gtggggtaaa attggaatca ataggaactt accaaatact gtcaatttat 1560 tcaacagtgg cgagttccct agcactggca atcatgatgg ctggtctatc tttatggatg 1620 tgctccaatg gatcgttaca atgcagaatt tgcatttaa 1659 <210> 8 <211> 1729 <212> DNA <213> Influenza A virus (A/Indonesia/05/2005 (H5N1)) <400> 8 ctgtaaaaat ggagaaaata gtgcttcttc ttgcaatagt cagtcttgtt aaaagtgatc 60 agatttgcat tggttaccat gcaaacaatt caacagagca ggttgacaca atcatggaaa 120 agaacgttac tgttacacat gcccaagaca tactggaaaa gacacacaac gggaagctct 180 gcgatctaga tggagtgaag cctctaattt taagagattg tagtgtagct ggatggctcc 240 tcgggaaccc aatgtgtgac gaattcatca atgtaccgga atggtcttac atagtggaga 300 aggccaatcc aaccaatgac ctctgttacc cagggagttt caacgactat gaagaactga 360 aacacctatt gagcagaata aaccattttg agaaaattca aatcatcccc aaaagttctt 420 ggtccgatca tgaagcctca tcaggagtga gctcagcatg tccatacctg ggaagtccct 480 ccttttttag aaatgtggta tggcttatca aaaagaacag tacataccca acaataaaga 540 aaagctacaa taataccaac caagaagatc ttttggtact gtggggaatt caccatccta 600 atgatgcggc agagcagaca aggctatatc aaaacccaac cacctatatt tccattggga 660 catcaacact aaaccagaga ttggtaccaa aaatagctac tagatccaaa gtaaacgggc 720 aaagtggaag gatggagttc ttctggacaa ttttaaaacc taatgatgca atcaacttcg 780 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag aaaggggact 840 cagcaattat gaaaagtgaa ttggaatatg gtaactgcaa caccaagtgt caaactccaa 900 tgggggcgat aaactctagt atgccattcc acaacataca ccctctcacc atcggggaat 960 gccccaaata tgtgaaatca aacagattag tccttgcaac agggctcaga aatagccctc 1020 aaagagagag cagaagaaaa aagagaggac tatttggagc tatagcaggt tttatagagg 1080 gaggatggca gggaatggta gatggttggt atgggtacca ccatagcaat gagcagggga 1140 gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc accaataagg 1200 tcaactcaat cattgacaaa atgaacactc agtttgaggc cgttggaagg gaatttaata 1260 acttagaaag gagaatagag aatttaaaca agaagatgga agacgggttt ctagatgtct 1320 ggacttataa tgccgaactt ctggttctca tggaaaatga gagaactcta gactttcatg 1380 actcaaatgt taagaacctc tacgacaagg tccgactaca gcttagggat aatgcaaagg 1440 agctgggtaa cggttgtttc gagttctatc acaaatgtga taatgaatgt atggaaagta 1500 taagaaacgg aacgtacaac tatccgcagt attcagaaga agcaagatta aaaagagagg 1560 aaataagtgg ggtaaaattg gaatcaatag gaacttacca aatactgtca atttattcaa 1620 cagtggcgag ttccctagca ctggcaatca tgatggctgg tctatcttta tggatgtgct 1680 ccaatggatc gttacaatgc agaatttgca tttaaatttg tgagttcag 1729 <210> 9 <211> 1673 <212> DNA <213> Influenza A virus (A/Chicken/Korea/ES/2003(H5N1)) <400> 9 gatcagattt gcattggtta ccatgcaaac aactcgacag agcaggttga cacaataatg 60 gaaaagaacg tcactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 120 ctctgcgatc tagatggagt gaagcctcta attttaagag attgtagtgt agctggatgg 180 ctcctcggga acccaatgtg tgacgaattc atcaatgtgc cggaatggtc ttacatagtg 240 gagaaggcca atccacccaa tgacctctgt tacccaggga atttcaacga ctatgaagaa 300 ctgaaacacc tattgagcag aataaaccat tttgaaaaaa ttcagatcat ccccaaaagt 360 tcttggtccg atcatgaagc ctcatcaggg gtgagctcag catgtccata ccagggaagg 420 tcctccttct tcagaaatgt ggtatggctt atcaaaaaga acagtgcata cccaacaata 480 aagagaagct acaataatac caaccaagaa gatcttttgg tactgtgggg gattcaccat 540 ccaaatgatg cggcagagca gacaagactc tatcaaaacc caaccaccta tatttccgtt 600 gggacatcaa cactaaacca gagattggta ccaaaaatag ctactagatc caaagtaaac 660 gggcaaagtg gaaggatgga gttcttctgg acaattttaa aaccgaatga tgcaatcagc 720 tttgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 780 gactcagcaa ttatgaaaag tgaattggaa tatggtaact gcaacaccaa gtgtcaaact 840 ccaatggggg cgataaactc tagtatgcca ttccacaaca tacaccctct caccatcggg 900 gaatgcccca aatatgtgaa atcaagcaga ttagtccttg cgactgggct cagaaatagc 960 cctcaaagag agnnnaaaag aaaaaagaga ggactatttg gagctatagc aggttttata 1020 gagggaggat ggcagggaat ggtagatggt tggtatgggt accaccatag caatgagcag 1080 gggagtgggt acgctgcaga caaagaatcc actcaaaagg caatagatgg agtcaccaat 1140 aaggtcaact cgatcattga caaaatgaac actcagtttg aggccgttgg aagggaattt 1200 aataacttag aaaggagaat agaaaattta aacaagaaga tggaagacgg attcctagat 1260 gtctggactt ataatgctga acttctggtt ctcatggaaa atgagagaac tctagacttt 1320 catgactcaa atgtcaagaa cctttacgac aaggtccgac tacagcttag ggataatgca 1380 aaggagctgg gtaacggttg tttcgagttc tatcacagat gtgataatga atgtatagaa 1440 agtgtaagaa acggaacgta tgactacccg cagtattcag aagaagcaag attaaaaaga 1500 gaggaaataa gtggagtaaa attggaatca ataggaactt accaaatact gtcaatttat 1560 tcaacagtgg caagttccct agcactggca atcatggtgg ctggtctatc tttatggatg 1620 tgctccaatg gatcgttaca atgcagaatt tgcatttaaa tttgtgagtt cag 1673 <210> 10 <211> 1779 <212> DNA <213> Influenza A virus(A/Vietnam/1203/2004 (H5N1)) <400> 10 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt 60 cagtcttgtt aaaagtgatc agatttgcat tggttaccat gcaaacaact cgacagagca 120 ggttgacaca ataatggaaa agaacgttac tgttacacat gcccaagaca tactggaaaa 180 gaaacacaac gggaagctct gcgatctaga tggagtgaag cctctaattt tgagagattg 240 tagcgtagct ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc caggggattt 360 caatgactat gaagaattga aacacctatt gagcagaata aaccattttg agaaaattca 420 gatcatcccc aaaagttctt ggtccagtca tgaagcctca ttaggggtga gctcagcatg 480 tccataccag ggaaagtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540 tacataccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc aaaacccaac 660 cacctatatt tccgttggga catcaacact aaaccagaga ttggtaccaa gaatagctac 720 tagatccaaa gtaaacgggc aaagtggaag gatggagttc ttctggacaa ttttaaagcc 780 gaatgatgca atcaacttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc acaatataca 960 ccctctcacc attggggaat gccccaaata tgtgaaatca aacagattag tccttgcgac 1020 tgggctcaga aatagccctc aaagagagag aagaagaaaa aagagaggat tatttggagc 1080 tatagcaggt tttatagagg gaggatggca gggaatggta gatggttggt atgggtacca 1140 ccatagcaat gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat 1200 agatggagtc accaataagg tcaactcgat cattgacaaa atgaacactc agtttgaggc 1260 cgttggaagg gaatttaaca acttagaaag gagaatagag aatttaaaca agaagatgga 1320 agacgggttc ctagatgtct ggacttataa tgctgaactt ctggttctca tggaaaatga 1380 gagaactcta gactttcatg actcaaatgt caagaacctt tacgacaagg tccgactaca 1440 gcttagggat aatgcaaagg agctgggtaa cggttgtttc gagttctatc ataaatgtga 1500 taatgaatgt atggaaagtg taagaaatgg aacgtatgac tacccgcagt attcagaaga 1560 agcgagacta aaaagagagg aaataagtgg agtaaaattg gaatcaatag gaatttacca 1620 aatactgtca atttattcta cagtggcgag ttccctagca ctggcaatca tggtagctgg 1680 tctatcctta tggatgtgct ccaatgggtc gttacaatgc agaatttgca tttaaatttg 1740 tgagttcaga ttgtagttaa aaacaccctt gtttctact 1779 <210> 11 <211> 1704 <212> DNA <213> Influenza A virus(A/Vietnam/HN31242/2007(H5N1)) <400> 11 atggagaaaa tagtgcttct tcttgcaata atcagccttg ttaaaagtga tcagatttgc 60 attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120 actgttacac atgctcaaga tatactggaa aagacacaca acgggaagct ctgcgatcta 180 gatggagtga agcctctgat tttaagagat tgtagtgtag ctggatggct cctcggaaac 240 ccaatgtgtg acgaattcat caatgtgccg gaatggtctt acatagtgga gaaggccaac 300 ccagccaatg acctctgtta cccaggaaat ttcaacgact atgaagaact gaaacaccta 360 ttgagcagga taaaccattt tgagaaaatt cagatcatcc ccaaaagttc ttggtccgat 420 catgaagcct catcaggggt gagctcagca tgtccatacc agggagtgcc ctcctttttc 480 agaaatgtgg tatggcttat caaaaagaac aatacatacc caacaataaa gagaagctac 540 aataatacca accaggaaga tcttttgata ctgtggggga ttcatcattc taatgatgca 600 gcagagcaga caaagctcta tcaaaaccca accacctata tttccgttgg gacatcaaca 660 ctgaaccaga gattggtacc aaaaatagct actagatcca aagtaaacgg gcaaagcgga 720 aggatggatt tcttctggac aattttaaaa ccgaatgatg caatcaactt cgagagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcggcaatt 840 atgaaaagtg aggtggagta tggtaactgc aacaccaagt gtcaaactcc aataggggcg 900 ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa 960 tatgtgaaat caaacaaatt agtccttgcg actggactca gaaatagtcc tctaagagaa 1020 agaagaagaa aaagaggact atttggagct atagcaggtt ttatagaggg cggatggcag 1080 ggaatggtag atggttggta tgggtatcac catagcaatg agcaggggag tgggtacgct 1140 gcagacaaag aatccactca aaaggcaata gatggagtca ccaataaggt caactcaatc 1200 attgacaaaa tgaacactca gtttgaggcc gttggaaggg aatttaataa cttagagagg 1260 agaatagaga atttaaacaa gaaaatggaa gacggattcc tagatgtctg gacttataat 1320 gctgaacttc tggttctcat ggaaaatgag agaactctag acttccatga ttcaaatgtc 1380 aagaaccttt acgacaaggt ccgactacag cttagggata atgcaaagga gctgggtaac 1440 ggttgtttca agttctatca caaatgtgat aatgaatgca tggaaagtgt aagaaacgga 1500 acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560 gtaaaattgg aatcaatagg aacttaccaa atactgtcaa tttattcaac agttgcgagt 1620 tctctagcac tggcaatcat ggtggctggt ctatctttat ggatgtgctc caatgggtcg 1680 ttacaatgca gaattcgcat ttaa 1704 <210> 12 <211> 1350 <212> DNA <213> Influenza A virus(A/Indonesia/05/2005(H5N1)) <400> 12 atgaatccaa atcagaagat aataaccatt ggatcaatct gtatggtaat tggaatagtt 60 agcttaatgt tacaaattgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaac accaagctga atcaatcagc aatactaacc ctcttactga gaaagctgtg 180 gcttcagtaa cattagcggg caattcatct ctttgcccca ttagaggatg ggctgtacac 240 agtaaggaca acaatataag gatcggttcc aagggggatg tgtttgttat tagagagccg 300 ttcatctcat gctcccacct ggaatgcaga actttcttct tgactcaggg agccttgctg 360 aatgacaagc actccaacgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctctccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccagacaat 540 gaggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgtg taaatggctc ttgctttact 660 gtaatgactg atggaccaag tgatgggcag gcatcatata agatcttcaa aatggaaaaa 720 ggaaaagtgg tcaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtttgcaggg ataattggca tggctcaaat 840 aggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtgga 900 gttttcggag acaatccacg ccccaatgat ggaacaggta gttgtggccc gatgtcccct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actggaacgg acagtagctt ttcagtgaaa caagatatag tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gattagattg cataagacct 1200 tgtttctggg ttgagttaat cagagggcgg cccaaagaga gcacaatttg gactagtggg 1260 agcagcatat ctttttgtgg tgtaaatagt gacactgtga gttggtcttg gccagacggt 1320 gctgagttgc cattcaccat tgacaagtag 1350 <210> 13 <211> 1388 <212> DNA <213> Influenza A virus(A/Indonesia/05/2005(H5N1)) <400> 13 gagattaaaa tgaatccaaa tcagaagata ataaccattg gatcaatctg tatggtaatt 60 ggaatagtta gcttaatgtt acaaattggg aacatgatct caatatgggt cagtcattca 120 attcagacag ggaatcaaca ccaagctgaa tcaatcagca atactaaccc tcttactgag 180 aaagctgtgg cttcagtaac attagcgggc aattcatctc tttgccccat tagaggatgg 240 gctgtacaca gtaaggacaa caatataagg atcggttcca agggggatgt gtttgttatt 300 agagagccgt tcatctcatg ctcccacctg gaatgcagaa ctttcttctt gactcaggga 360 gccttgctga atgacaagca ctccaacggg actgtcaaag acagaagccc tcacagaaca 420 ttaatgagtt gtcctgtggg tgaggctccc tctccatata actcaaggtt tgagtctgtt 480 gcttggtcag caagtgcttg ccatgatggc accagttggt tgacaattgg aatttctggc 540 ccagacaatg aggctgtggc tgtattgaaa tacaatggca taataacaga cactatcaag 600 agttggagga acaacatact gagaactcaa gagtctgaat gtgcatgtgt aaatggctct 660 tgctttactg taatgactga tggaccaagt gatgggcagg catcatataa gatcttcaaa 720 atggaaaaag gaaaagtggt caaatcagtc gaattggatg ctcctaatta tcactatgag 780 gaatgctcct gttatcctga tgccggcgaa atcacatgtg tttgcaggga taattggcat 840 ggctcaaata ggccatgggt atctttcaat caaaatttgg agtatcaaat aggatatata 900 tgcagtggag ttttcggaga caatccacgc cccaatgatg gaacaggtag ttgtggcccg 960 atgtccccta acggggcata tggggtaaaa gggttttcat ttaaatacgg caatggtgtt 1020 tggatcggga gaaccaaaag cactaattcc aggagcggct ttgaaatgat ttgggatcca 1080 aatgggtgga ctggaacgga cagtagcttt tcagtgaaac aagatatagt agcaataact 1140 gattggtcag gatatagcgg gagttttgtc cagcatccag aactgacagg attagattgc 1200 ataagacctt gtttctgggt tgagttaatc agagggcggc ccaaagagag cacaatttgg 1260 actagtggga gcagcatatc tttttgtggt gtaaatagtg acactgtgag ttggtcttgg 1320 ccagacggtg ctgagttgcc attcaccatt gacaagtagt ttgttcaaaa aatcctttgg 1380 ttttctac 1388 <210> 14 <211> 1350 <212> DNA <213> Influenza A virus(A/chicken/Korea/ES/2003(H5N1)) <400> 14 atgaatccaa atcagaagat aataaccatc ggatcaatct gtatggtaat tggaatagtt 60 agcttaatgt tacaagttgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaac gcaaagctga accaatcagc aatactaaat ttcttactga gaaagctgtg 180 acttcagtaa cattagcggg caattcatct ctttgcccca ttagcggatg ggctgtacac 240 agtaaggaca acagtataag gatcggttcc aagggggatg tgtttgttat aagagagccg 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaatgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccagacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgcg taaatggctc ttgctttact 660 gtaatgactg atggaccaag taatgggcag gcatcatata agatcttcaa aatggaaaaa 720 ggaaaagtgg ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtgtgcaggg ataattggca tggctcaaat 840 aggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtggg 900 gttttcggag acaatccacg ccccaatgat ggaacaggta gttgtggtcc ggtgtcccct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actggaacgg acagtagctt ttcggtgaag caagatatcg tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gattagattg cataagacct 1200 tgtttctggg ttgagttaat cagagggcgg cccaaggaga gcacaatttg gactagtggg 1260 agcagcatat ccttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1320 gctgagttgc cattcaccat tgacaagtag 1350 <210> 15 <211> 1350 <212> DNA <213> Influenza A virus(A/Vietnam/1203/2004(H5N1)) <400> 15 atgaatccaa atcagaagat aataaccatc ggatcaatct gtatggtaac tggaatagtt 60 agcttaatgt tacaaattgg gaacatgatc tcaatatggg tcagtcattc aattcacaca 120 gggaatcaac accaatctga accaatcagc aatactaatt ttcttactga gaaagctgtg 180 gcttcagtaa aattagcggg caattcatct ctttgcccca ttaacggatg ggctgtatac 240 agtaaggaca acagtataag gatcggttcc aagggggatg tgtttgttat aagagagccg 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaatgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacgattg gaatttctgg cccagacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgtg taaatggctc ttgctttact 660 gtaatgactg acggaccaag taatggtcag gcatcacata agatcttcaa aatggaaaaa 720 gggaaagtgg ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatccta atgccggaga aatcacatgt gtgtgcaggg ataattggca tggctcaaat 840 cggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtgga 900 gttttcggag acaatccacg ccccaatgat ggaacaggta gttgtggtcc ggtgtcctct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ctggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actgaaacgg acagtagctt ttcagtgaaa caagatatcg tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gactagattg cataagacct 1200 tgtttctggg ttgagttgat cagagggcgg cccaaagaga gcacaatttg gactagtggg 1260 agcagcatat ctttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1320 gccgagttgc cattcaccat tgacaagtag 1350 <210> 16 <211> 1350 <212> DNA <213> Influenza A virus(A/Vietnam/HN31242/2007(H5N1)) <400> 16 atgaatccaa atcagaagat aataaccatt gggtcaatct gtatggtaat tggaatagtt 60 agcttaatgt tgcaaattgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaaa accaagttga gccaatcatc aatactaatt ttcttactgg gaaagctgtg 180 gcttcaataa cattagcggg caattcatct ctttgcccta ttagaggatg ggctatacac 240 agtaaggaca acagtataag gattggttcc aagggggatg tgtttgttat tagagagcca 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaacgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccggacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acaccatcaa gagctggagg 600 aacaacatac tgagaactca agagtccgaa tgtgcatgcg taaatggctc ttgctttact 660 gtaatgactg acggaccaag taatgggcag gcatcatata agatcttcaa aatggaaaag 720 gggaaagtaa ttaaatcagt cgaattggat gctcctaatt accactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtgtgcagag ataattggca tggctcaaat 840 cggccatggg tgtccttcaa tcaaaatctg gagtatcaaa taggatatat atgcagtgga 900 gttttcggag acaatccacg ccccaatgat ggaacaggta gttgcggtcc ggtgtcccat 960 aatggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gccctaattc caggagcggc tttgagatga tttgggatcc aaatgggtgg 1080 agtgaaacgg acagtagctt ctcggtgaaa caagatatag tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gtttagattg cataagacct 1200 tgtttctggg ttgaattaat cagagggcgg cccaaagaga gtacagtttg gactagtggg 1260 agcagcatat ccttttgtgg tgtaaatagt gacactgtga gttggtcttg gccagacggt 1320 gctgagttgc cattcagcat tgacaagtag 1350 <110> Industry-Academic Cooperation Foundation, Yonsei University <120> Method for preparing cold adapted to live vaccine of          highly pathogenic avian influenza H5N1 <130> IPDB46147 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 2316 <212> DNA <213> x-31 ca virus_PB2 <400> 1 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg aaatctaatg 60 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc 120 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg 180 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat 240 gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta 300 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcagtat 360 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc 420 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat 480 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa 600 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg 660 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg 720 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg 780 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca 840 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga 900 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc 960 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag 1020 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggaaa tcttcaaaca 1080 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca 1140 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa 1200 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata 1260 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcagcgatt gaatcctatg 1320 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt 1380 gaacctatcg acaatgtgat gggaatgatt gggatattgc ccgacatgac tccaagcatc 1440 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg 1500 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa 1680 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta 1740 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccag tagaggccaa 1800 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg 1920 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat 2040 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg 2100 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat 2160 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg 2220 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280 aaaagaattc ggatggccat caattagtgt tgaata 2316 <210> 2 <211> 2341 <212> DNA <213> x-31 ca virus_PB1 <400> 2 agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60 ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat 120 gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag 180 ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca 240 ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt ggaggcgatg 300 gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgtattga aacgatggag 360 gttgttcagc aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact 420 ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt gttcagatca 480 aatggcctca cggccaatga gtctggaagg ctcatagact tccttaagga tgtaatggag 540 tcaatgaaca aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga 600 gacaatatga ctaagaaaat gataacacag agaacaatgg gtaaaaagaa gcagagattg 660 aacaaaagga gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag 720 agagggaagc taaaacggag aacaattgca accccaggga tgcaaataag ggggtttgta 780 tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca 840 gtggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat gatgaccaat 900 tctcaggaca ccgaactttc tttcaccatc actggagata acaccaaatg gaacgaaaat 960 cagaatcctc ggatgttttt ggccatgatc acatatatga ccagaaatca gcccgaatgg 1020 ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga 1080 aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg 1140 ctagcaagca tcgatttgaa atatttcaat gattcaacaa gaaagaagat tgaaaaaatc 1200 cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260 aatatgttga gcactgtatt aggcgtctcc atcctgaacc ttggacaaaa gagatacacc 1320 aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat 1380 gcacccaatc atgaagggat tcaagccgga gtcgacaggt tttatcgaac ctgtaagcta 1440 cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc 1500 acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct tcccagtttt 1560 ggggtgtctg ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac 1620 aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc 1680 aaagattaca ggtacacgta ccgatgccat agaggtgaca cacaaataca aacccgaaga 1740 tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc 1800 gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860 tgggaattga tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc 1920 agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca tggtccagcc 1980 aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatcga 2040 tccatcttga atacaagtca aagaggagta cttgaggatg aacaaatgta ccaaaggtgc 2100 tgcaatttat ttgaaaaatt cttccccagc agttcataca gaagaccagt cgggatatcc 2160 agtatggtgg aggctatggt ttctagagcc cgaattgatg cacggattga tttcgaatct 2220 ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag 2280 ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340 t 2341 <210> 3 <211> 2233 <212> DNA <213> x-31 ca virus_PA <400> 3 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca 120 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac 180 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg 240 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac 300 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac 360 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg 420 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg 480 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa 540 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt 600 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc 660 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat 720 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa 780 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat 840 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt 900 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga 960 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca 1020 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag 1140 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa 1200 tatgatagtg atgaaccaga attgaggtca cttgcaagtt ggattcagaa tgagtttaac 1260 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg 1320 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac 1380 tgcagagcca cagaatacat aatgaaggga gtgtacatca atactgcctt gcttaatgca 1440 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag 1500 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt 1680 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa 1740 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt 1800 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc 1920 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct 1980 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttgtcgt tcaggctctt 2040 agggacaacc tggaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta 2220 ccttgtttct act 2233 <210> 4 <211> 1565 <212> DNA <213> x-31 ca virus_NP <400> 4 agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtctcaaggc 60 accaaacgat cttacgaaca gatggagact gatggaggac gccagaatgc cactgaaatc 120 agagcatccg tcggaaaaat gattggtgga attggacgat tctacatcca aatgtgcacc 180 gaactcaaac tcagtgatta tgagggacgg ttgatccaaa acagcttaac aatagagaga 240 atggtgctct ctgcttttga cgaaaggaga aataaatacc ttgaagaaca tcccagtgcg 300 gggaaagatc ctaagaaaac tggaggacct atatacagga gagtaaacgg aaagtggatg 360 agagaactca tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat 420 ggtgacgatg caatggctgg tctgactcac atgatgatct ggcattccaa tttgaatgat 480 gcaacttatc agaggacaag agctcttgtt cgcaccggaa tggatcccag gatgtgctct 540 ctgatgcaag gttcaactct ccctaggagg tctggagccg caggtgctgc agtcaaagga 600 gttggaacaa tggtgatgga attggtcaga atgatcaaac gtgggatcaa tgatcggaac 660 ttctggaggg gtgagaatgg acgaaaaaca agaattgctt atgaaagaat gtgcaacatt 720 ctcaaaggga aatttcaaac tgctgcacaa aaagcaatga tggatcaagt gagagagagc 780 cggaacccag ggaatgctga gttcgaagat ctcacttttc tagcacggtc tgcactcata 840 ttgagagggt cggttgctca caagtcctgc ctgcctgcct gtgtgtatgg acctgccgta 900 gccagtgggt acgactttga aagggaggga tactctctag tcggaataga ccctttcaga 960 ctgcttcaaa acagccaagt gtacagccta atcagaccaa atgagaatcc agcacacaag 1020 agtcaactgg tgtggatggc atgccattct gccgcatttg aagatctaag agtattaagc 1080 ttcatcaaag ggacgaaggt gctcccaaga gggaagcttt ccactagagg agttcaaatt 1140 gcttccaatg aaaatatgga gactatggaa tcaagtacac ttgaactgag aagcaggtac 1200 tgggccataa ggaccagaag tggaggaaac accaatcaac agagggcatc tgcgggccaa 1260 atcagcatac aacctacgtt ctcagtacag agaaatctcc cttttgacag aaccaccatt 1320 atggcagcat tcaatgggaa tacagagggg agaacatctg acatgaggac cgaaatcata 1380 aggatgatgg aaagtgcaag accagaagat gtgtctttcc aggggcgggg agtcttcgag 1440 ctctcggacg aaaaggcagc gagcccgatc gtgccttcct ttgacatgag taatgaagga 1500 tcttatttct tcggagacaa tgcagaggag tacgacaatt aaagaaaaat acccttgttt 1560 ctact 1565 <210> 5 <211> 1027 <212> DNA <213> x-31 ca virus_M <400> 5 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact 60 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120 tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420 caacaggatg gggactgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact 540 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat 660 ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga 720 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780 gtgatcctct cactattgcc gcaaatatca ttgggatttt gcacttgaca ttgtggattc 840 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020 ttctact 1027 <210> 6 <211> 890 <212> DNA <213> x-31 ca virus_NS <400> 6 agcaaaagca gggtgacaaa aacataatgg atccaaacac tgtgtcaagc tttcaggtag 60 attgctttct ttggcatgtc cgcaaacgag ttgcagacca agaactaggt gatgccccat 120 tccttgatcg gcttcgccga gatcagaaat ccctaagagg aaggggcagt actctcggtc 180 tggacatcaa gacagccaca cgtgctggaa agcagatagt ggagcggatt ctgaaagaag 240 aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt tacctaactg 300 acatgactct tgaggaattg tcaagggact ggtccatgct catacccaag cagaaagtgg 360 caggccctct ttgtatcaga atggaccagg cgatcatgga taagaacatc atactgaaag 420 cgaacttcag tgtgattttt gaccggctgg agactctaat attgctaagg gctttcaccg 480 aagagggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactgctg 540 aggatgtcaa aaatgcagtt ggagtcctca tcggaggact tgaatggaat gataacacag 600 ttcgagtctc tgaaactcta cagagattcg cttggagaag cagtaatgag aatgggagac 660 ctccactcac tccaaaacag aaacgagaaa tggcgggaac aattaggtca gaagtttgaa 720 gaaataagat ggttgattga agaagtgaga cacaaactga agataacaga gaatagtttt 780 gagcaaataa catttatgca agccttacat ctattgcttg aagtggagca agagataaga 840 actttctcgt ttcagcttat ttagtactaa aaaacaccct tgtttctact 890 <210> 7 <211> 1659 <212> DNA <213> Influenzae virus (A / Indonesia / 05/2005 (H5N1)) <400> 7 gatcagattt gcattggtta ccatgcaaac aattcaacag agcaggttga cacaatcatg 60 gaaaagaacg ttactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 120 ctctgcgatc tagatggagt gaagcctcta attttaagag attgtagtgt agctggatgg 180 ctcctcggga acccaatgtg tgacgaattc atcaatgtac cggaatggtc ttacatagtg 240 gagaaggcca atccaaccaa tgacctctgt tacccaggga gtttcaacga ctatgaagaa 300 ctgaaacacc tattgagcag aataaaccat tttgagaaaa ttcaaatcat ccccaaaagt 360 tcttggtccg atcatgaagc ctcatcagga gtgagctcag catgtccata cctgggaagt 420 ccctcctttt ttagaaatgt ggtatggctt atcaaaaaga acagtacata cccaacaata 480 aagaaaagct acaataatac caaccaagaa gatcttttgg tactgtgggg aattcaccat 540 cctaatgatg cggcagagca gacaaggcta tatcaaaacc caaccaccta tatttccatt 600 gggacatcaa cactaaacca gagattggta ccaaaaatag ctactagatc caaagtaaac 660 gggcaaagtg gaaggatgga gttcttctgg acaattttaa aacctaatga tgcaatcaac 720 ttcgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 780 gactcagcaa ttatgaaaag tgaattggaa tatggtaact gcaacaccaa gtgtcaaact 840 ccaatggggg cgataaactc tagtatgcca ttccacaaca tacaccctct caccatcggg 900 gaatgcccca aatatgtgaa atcaaacaga ttagtccttg caacagggct cagaaatagc 960 cctcaaagag agagcagaag aaaaaagaga ggactatttg gagctatagc aggttttata 1020 gagggaggat ggcagggaat ggtagatggt tggtatgggt accaccatag caatgagcag 1080 gggagtgggt acgctgcaga caaagaatcc actcaaaagg caatagatgg agtcaccaat 1140 aaggtcaact caatcattga caaaatgaac actcagtttg aggccgttgg aagggaattt 1200 aataagtag aaaggagaat agagaattta aacaagaaga tggaagacgg gtttctagat 1260 gtctggactt ataatgccga acttctggtt ctcatggaaa atgagagaac tctagacttt 1320 catgactcaa atgttaagaa cctctacgac aaggtccgac tacagcttag ggataatgca 1380 aaggagctgg gtaacggttg tttcgagttc tatcacaaat gtgataatga atgtatggaa 1440 agtataagaa acggaacgta caactatccg cagtattcag aagaagcaag attaaaaaga 1500 gaggaaataa gtggggtaaa attggaatca ataggaactt accaaatact gtcaatttat 1560 tcaacagtgg cgagttccct agcactggca atcatgatgg ctggtctatc tttatggatg 1620 tgctccaatg gatcgttaca atgcagaatt tgcatttaa 1659 <210> 8 <211> 1729 <212> DNA <213> Influenzae virus (A / Indonesia / 05/2005 (H5N1)) <400> 8 ctgtaaaaat ggagaaaata gtgcttcttc ttgcaatagt cagtcttgtt aaaagtgatc 60 agatttgcat tggttaccat gcaaacaatt caacagagca ggttgacaca atcatggaaa 120 agaacgttac tgttacacat gcccaagaca tactggaaaa gacacacaac gggaagctct 180 gcgatctaga tggagtgaag cctctaattt taagagattg tagtgtagct ggatggctcc 240 tcgggaaccc aatgtgtgac gaattcatca atgtaccgga atggtcttac atagtggaga 300 aggccaatcc aaccaatgac ctctgttacc cagggagttt caacgactat gaagaactga 360 aacacctatt gagcagaata aaccattttg agaaaattca aatcatcccc aaaagttctt 420 ggtccgatca tgaagcctca tcaggagtga gctcagcatg tccatacctg ggaagtccct 480 ccttttttag aaatgtggta tggcttatca aaaagaacag tacataccca acaataaaga 540 aaagctacaa taataccaac caagaagatc ttttggtact gtggggaatt caccatccta 600 atgatgcggc agagcagaca aggctatatc aaaacccaac cacctatatt tccattggga 660 catcaacact aaaccagaga ttggtaccaa aaatagctac tagatccaaa gtaaacgggc 720 aaagtggaag gatggagttc ttctggacaa ttttaaaacc taatgatgca atcaacttcg 780 agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag aaaggggact 840 cagcaattat gaaaagtgaa ttggaatatg gtaactgcaa caccaagtgt caaactccaa 900 tgggggcgat aaactctagt atgccattcc acaacataca ccctctcacc atcggggaat 960 gccccaaata tgtgaaatca aacagattag tccttgcaac agggctcaga aatagccctc 1020 aaagagagag cagaagaaaa aagagaggac tatttggagc tatagcaggt tttatagagg 1080 gaggatggca gggaatggta gatggttggt atgggtacca ccatagcaat gagcagggga 1140 gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc accaataagg 1200 tcaactcaat cattgacaaa atgaacactc agtttgaggc cgttggaagg gaatttaata 1260 acttagaaag gagaatagag aatttaaaca agaagatgga agacgggttt ctagatgtct 1320 ggacttataa tgccgaactt ctggttctca tggaaaatga gagaactcta gactttcatg 1380 actcaaatgt taagaacctc tacgacaagg tccgactaca gcttagggat aatgcaaagg 1440 agctgggtaa cggttgtttc gagttctatc acaaatgtga taatgaatgt atggaaagta 1500 taagaaacgg aacgtacaac tatccgcagt attcagaaga agcaagatta aaaagagag 1560 aaataagtgg ggtaaaattg gaatcaatag gaacttacca aatactgtca atttattcaa 1620 cagtggcgag ttccctagca ctggcaatca tgatggctgg tctatcttta tggatgtgct 1680 ccaatggatc gttacaatgc agaatttgca tttaaatttg tgagttcag 1729 <210> 9 <211> 1673 <212> DNA <213> Influenzae virus (A / Chicken / Korea / ES / 2003 (H5N1)) <400> 9 gatcagattt gcattggtta ccatgcaaac aactcgacag agcaggttga cacaataatg 60 gaaaagaacg tcactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 120 ctctgcgatc tagatggagt gaagcctcta attttaagag attgtagtgt agctggatgg 180 ctcctcggga acccaatgtg tgacgaattc atcaatgtgc cggaatggtc ttacatagtg 240 gagaaggcca atccacccaa tgacctctgt tacccaggga atttcaacga ctatgaagaa 300 ctgaaacacc tattgagcag aataaaccat tttgaaaaaa ttcagatcat ccccaaaagt 360 tcttggtccg atcatgaagc ctcatcaggg gtgagctcag catgtccata ccagggaagg 420 tcctccttct tcagaaatgt ggtatggctt atcaaaaaga acagtgcata cccaacaata 480 aagagaagct acaataatac caaccaagaa gatcttttgg tactgtgggg gattcaccat 540 ccaaatgatg cggcagagca gacaagactc tatcaaaacc caaccaccta tatttccgtt 600 gggacatcaa cactaaacca gagattggta ccaaaaatag ctactagatc caaagtaaac 660 gggcaaagtg gaaggatgga gttcttctgg acaattttaa aaccgaatga tgcaatcagc 720 tttgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 780 gactcagcaa ttatgaaaag tgaattggaa tatggtaact gcaacaccaa gtgtcaaact 840 ccaatggggg cgataaactc tagtatgcca ttccacaaca tacaccctct caccatcggg 900 gaatgcccca aatatgtgaa atcaagcaga ttagtccttg cgactgggct cagaaatagc 960 cctcaaagag agnnnaaaag aaaaaagaga ggactatttg gagctatagc aggttttata 1020 gagggaggat ggcagggaat ggtagatggt tggtatgggt accaccatag caatgagcag 1080 gggagtgggt acgctgcaga caaagaatcc actcaaaagg caatagatgg agtcaccaat 1140 aaggtcaact cgatcattga caaaatgaac actcagtttg aggccgttgg aagggaattt 1200 aataagtag aaaggagaat agaaaattta aacaagaaga tggaagacgg attcctagat 1260 gtctggactt ataatgctga acttctggtt ctcatggaaa atgagagaac tctagacttt 1320 catgactcaa atgtcaagaa cctttacgac aaggtccgac tacagcttag ggataatgca 1380 aaggagctgg gtaacggttg tttcgagttc tatcacagat gtgataatga atgtatagaa 1440 agtgtaagaa acggaacgta tgactacccg cagtattcag aagaagcaag attaaaaaga 1500 gaggaaataa gtggagtaaa attggaatca ataggaactt accaaatact gtcaatttat 1560 tcaacagtgg caagttccct agcactggca atcatggtgg ctggtctatc tttatggatg 1620 tgctccaatg gatcgttaca atgcagaatt tgcatttaaa tttgtgagtt cag 1673 <210> 10 <211> 1779 <212> DNA <213> Influenzae virus (A / Vietnam / 1203/2004 (H5N1)) <400> 10 agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt 60 cagtcttgtt aaaagtgatc agatttgcat tggttaccat gcaaacaact cgacagagca 120 ggttgacaca ataatggaaa agaacgttac tgttacacat gcccaagaca tactggaaaa 180 gaaacacaac gggaagctct gcgatctaga tggagtgaag cctctaattt tgagagattg 240 tagcgtagct ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc caggggattt 360 caatgactat gaagaattga aacacctatt gagcagaata aaccattttg agaaaattca 420 gatcatcccc aaaagttctt ggtccagtca tgaagcctca ttaggggtga gctcagcatg 480 tccataccag ggaaagtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540 tacataccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc aaaacccaac 660 cacctatatt tccgttggga catcaacact aaaccagaga ttggtaccaa gaatagctac 720 tagatccaaa gtaaacgggc aaagtggaag gatggagttc ttctggacaa ttttaaagcc 780 gaatgatgca atcaacttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc acaatataca 960 ccctctcacc attggggaat gccccaaata tgtgaaatca aacagattag tccttgcgac 1020 tgggctcaga aatagccctc aaagagagag aagaagaaaa aagagaggat tatttggagc 1080 tatagcaggt tttatagagg gaggatggca gggaatggta gatggttggt atgggtacca 1140 ccatagcaat gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat 1200 agatggagtc accaataagg tcaactcgat cattgacaaa atgaacactc agtttgaggc 1260 cgttggaagg gaatttaaca acttagaaag gagaatagag aatttaaaca agaagatgga 1320 agacgggttc ctagatgtct ggacttataa tgctgaactt ctggttctca tggaaaatga 1380 gagaactcta gactttcatg actcaaatgt caagaacctt tacgacaagg tccgactaca 1440 gcttagggat aatgcaaagg agctgggtaa cggttgtttc gagttctatc ataaatgtga 1500 taatgaatgt atggaaagtg taagaaatgg aacgtatgac tacccgcagt attcagaaga 1560 agcgagacta aaaagagagg aaataagtgg agtaaaattg gaatcaatag gaatttacca 1620 aatactgtca atttattcta cagtggcgag ttccctagca ctggcaatca tggtagctgg 1680 tctatcctta tggatgtgct ccaatgggtc gttacaatgc agaatttgca tttaaatttg 1740 tgagttcaga ttgtagttaa aaacaccctt gtttctact 1779 <210> 11 <211> 1704 <212> DNA <213> Influenzae virus (A / Vietnam / HN31242 / 2007 (H5N1)) <400> 11 atggagaaaa tagtgcttct tcttgcaata atcagccttg ttaaaagtga tcagatttgc 60 attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120 actgttacac atgctcaaga tatactggaa aagacacaca acgggaagct ctgcgatcta 180 gatggagtga agcctctgat tttaagagat tgtagtgtag ctggatggct cctcggaaac 240 ccaatgtgtg acgaattcat caatgtgccg gaatggtctt acatagtgga gaaggccaac 300 ccagccaatg acctctgtta cccaggaaat ttcaacgact atgaagaact gaaacaccta 360 ttgagcagga taaaccattt tgagaaaatt cagatcatcc ccaaaagttc ttggtccgat 420 catgaagcct catcaggggt gagctcagca tgtccatacc agggagtgcc ctcctttttc 480 agaaatgtgg tatggcttat caaaaagaac aatacatacc caacaataaa gagaagctac 540 aataatacca accaggaaga tcttttgata ctgtggggga ttcatcattc taatgatgca 600 gcagagcaga caaagctcta tcaaaaccca accacctata tttccgttgg gacatcaaca 660 ctgaaccaga gattggtacc aaaaatagct actagatcca aagtaaacgg gcaaagcgga 720 aggatggatt tcttctggac aattttaaaa ccgaatgatg caatcaactt cgagagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcggcaatt 840 atgaaaagtg aggtggagta tggtaactgc aacaccaagt gtcaaactcc aataggggcg 900 ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa 960 tatgtgaaat caaacaaatt agtccttgcg actggactca gaaatagtcc tctaagagaa 1020 agaagaagaa aaagaggact atttggagct atagcaggtt ttatagaggg cggatggcag 1080 ggaatggtag atggttggta tgggtatcac catagcaatg agcaggggag tgggtacgct 1140 gcagacaaag aatccactca aaaggcaata gatggagtca ccaataaggt caactcaatc 1200 attgacaaaa tgaacactca gtttgaggcc gttggaaggg aatttaataa cttagagagg 1260 agaatagaga atttaaacaa gaaaatggaa gacggattcc tagatgtctg gacttataat 1320 gctgaacttc tggttctcat ggaaaatgag agaactctag acttccatga ttcaaatgtc 1380 aagaaccttt acgacaaggt ccgactacag cttagggata atgcaaagga gctgggtaac 1440 ggttgtttca agttctatca caaatgtgat aatgaatgca tggaaagtgt aagaaacgga 1500 acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560 gtaaaattgg aatcaatagg aacttaccaa atactgtcaa tttattcaac agttgcgagt 1620 tctctagcac tggcaatcat ggtggctggt ctatctttat ggatgtgctc caatgggtcg 1680 ttacaatgca gaattcgcat ttaa 1704 <210> 12 <211> 1350 <212> DNA <213> Influenzae virus (A / Indonesia / 05/2005 (H5N1)) <400> 12 atgaatccaa atcagaagat aataaccatt ggatcaatct gtatggtaat tggaatagtt 60 agcttaatgt tacaaattgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaac accaagctga atcaatcagc aatactaacc ctcttactga gaaagctgtg 180 gcttcagtaa cattagcggg caattcatct ctttgcccca ttagaggatg ggctgtacac 240 agtaaggaca acaatataag gatcggttcc aagggggatg tgtttgttat tagagagccg 300 ttcatctcat gctcccacct ggaatgcaga actttcttct tgactcaggg agccttgctg 360 aatgacaagc actccaacgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctctccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccagacaat 540 gaggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgtg taaatggctc ttgctttact 660 gtaatgactg atggaccaag tgatgggcag gcatcatata agatcttcaa aatggaaaaa 720 ggaaaagtgg tcaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtttgcaggg ataattggca tggctcaaat 840 aggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtgga 900 gtttcggag acaatccacg ccccaatgat ggaacaggta gttgtggccc gatgtcccct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actggaacgg acagtagctt ttcagtgaaa caagatatag tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gattagattg cataagacct 1200 tgtttctggg ttgagttaat cagagggcgg cccaaagaga gcacaatttg gactagtggg 1260 agcagcatat ctttttgtgg tgtaaatagt gacactgtga gttggtcttg gccagacggt 1320 gctgagttgc cattcaccat tgacaagtag 1350 <210> 13 <211> 1388 <212> DNA <213> Influenzae virus (A / Indonesia / 05/2005 (H5N1)) <400> 13 gagattaaaa tgaatccaaa tcagaagata ataaccattg gatcaatctg tatggtaatt 60 ggaatagtta gcttaatgtt acaaattggg aacatgatct caatatgggt cagtcattca 120 attcagacag ggaatcaaca ccaagctgaa tcaatcagca atactaaccc tcttactgag 180 aaagctgtgg cttcagtaac attagcgggc aattcatctc tttgccccat tagaggatgg 240 gctgtacaca gtaaggacaa caatataagg atcggttcca agggggatgt gtttgttatt 300 agagagccgt tcatctcatg ctcccacctg gaatgcagaa ctttcttctt gactcaggga 360 gccttgctga atgacaagca ctccaacggg actgtcaaag acagaagccc tcacagaaca 420 ttaatgagtt gtcctgtggg tgaggctccc tctccatata actcaaggtt tgagtctgtt 480 gcttggtcag caagtgcttg ccatgatggc accagttggt tgacaattgg aatttctggc 540 ccagacaatg aggctgtggc tgtattgaaa tacaatggca taataacaga cactatcaag 600 agttggagga acaacatact gagaactcaa gagtctgaat gtgcatgtgt aaatggctct 660 tgctttactg taatgactga tggaccaagt gatgggcagg catcatataa gatcttcaaa 720 atggaaaaag gaaaagtggt caaatcagtc gaattggatg ctcctaatta tcactatgag 780 gaatgctcct gttatcctga tgccggcgaa atcacatgtg tttgcaggga taattggcat 840 ggctcaaata ggccatgggt atctttcaat caaaatttgg agtatcaaat aggatatata 900 tgcagtggag ttttcggaga caatccacgc cccaatgatg gaacaggtag ttgtggcccg 960 atgtccccta acggggcata tggggtaaaa gggttttcat ttaaatacgg caatggtgtt 1020 tggatcggga gaaccaaaag cactaattcc aggagcggct ttgaaatgat ttgggatcca 1080 aatgggtgga ctggaacgga cagtagcttt tcagtgaaac aagatatagt agcaataact 1140 gattggtcag gatatagcgg gagttttgtc cagcatccag aactgacagg attagattgc 1200 ataagacctt gtttctgggt tgagttaatc agagggcggc ccaaagagag cacaatttgg 1260 actagtggga gcagcatatc tttttgtggt gtaaatagtg acactgtgag ttggtcttgg 1320 ccagacggtg ctgagttgcc attcaccatt gacaagtagt ttgttcaaaa aatcctttgg 1380 ttttctac 1388 <210> 14 <211> 1350 <212> DNA <213> Influenza A virus (A / chicken / Korea / ES / 2003 (H5N1)) <400> 14 atgaatccaa atcagaagat aataaccatc ggatcaatct gtatggtaat tggaatagtt 60 agcttaatgt tacaagttgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaac gcaaagctga accaatcagc aatactaaat ttcttactga gaaagctgtg 180 acttcagtaa cattagcggg caattcatct ctttgcccca ttagcggatg ggctgtacac 240 agtaaggaca acagtataag gatcggttcc aagggggatg tgtttgttat aagagagccg 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaatgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccagacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgcg taaatggctc ttgctttact 660 gtaatgactg atggaccaag taatgggcag gcatcatata agatcttcaa aatggaaaaa 720 ggaaaagtgg ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtgtgcaggg ataattggca tggctcaaat 840 aggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtggg 900 gtttcggag acaatccacg ccccaatgat ggaacaggta gttgtggtcc ggtgtcccct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actggaacgg acagtagctt ttcggtgaag caagatatcg tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gattagattg cataagacct 1200 tgtttctggg ttgagttaat cagagggcgg cccaaggaga gcacaatttg gactagtggg 1260 agcagcatat ccttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1320 gctgagttgc cattcaccat tgacaagtag 1350 <210> 15 <211> 1350 <212> DNA <213> Influenzae virus (A / Vietnam / 1203/2004 (H5N1)) <400> 15 atgaatccaa atcagaagat aataaccatc ggatcaatct gtatggtaac tggaatagtt 60 agcttaatgt tacaaattgg gaacatgatc tcaatatggg tcagtcattc aattcacaca 120 gggaatcaac accaatctga accaatcagc aatactaatt ttcttactga gaaagctgtg 180 gcttcagtaa aattagcggg caattcatct ctttgcccca ttaacggatg ggctgtatac 240 agtaaggaca acagtataag gatcggttcc aagggggatg tgtttgttat aagagagccg 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaatgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacgattg gaatttctgg cccagacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600 aacaacatac tgagaactca agagtctgaa tgtgcatgtg taaatggctc ttgctttact 660 gtaatgactg acggaccaag taatggtcag gcatcacata agatcttcaa aatggaaaaa 720 gggaaagtgg ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc 780 tgttatccta atgccggaga aatcacatgt gtgtgcaggg ataattggca tggctcaaat 840 cggccatggg tatctttcaa tcaaaatttg gagtatcaaa taggatatat atgcagtgga 900 gtttcggag acaatccacg ccccaatgat ggaacaggta gttgtggtcc ggtgtcctct 960 aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ctggatcggg 1020 agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080 actgaaacgg acagtagctt ttcagtgaaa caagatatcg tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacag gactagattg cataagacct 1200 tgtttctggg ttgagttgat cagagggcgg cccaaagaga gcacaatttg gactagtggg 1260 agcagcatat ctttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1320 gccgagttgc cattcaccat tgacaagtag 1350 <210> 16 <211> 1350 <212> DNA <213> Influenzae virus (A / Vietnam / HN31242 / 2007 (H5N1)) <400> 16 atgaatccaa atcagaagat aataaccatt gggtcaatct gtatggtaat tggaatagtt 60 agcttaatgt tgcaaattgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120 gggaatcaaa accaagttga gccaatcatc aatactaatt ttcttactgg gaaagctgtg 180 gcttcaataa cattagcggg caattcatct ctttgcccta ttagaggatg ggctatacac 240 agtaaggaca acagtataag gattggttcc aagggggatg tgtttgttat tagagagcca 300 ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360 aatgacaagc actccaacgg gactgtcaaa gacagaagcc ctcacagaac attaatgagt 420 tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480 gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccggacaat 540 ggggctgtgg ctgtattgaa atacaatggc ataataacag acaccatcaa gagctggagg 600 aacaacatac tgagaactca agagtccgaa tgtgcatgcg taaatggctc ttgctttact 660 gtaatgactg acggaccaag taatgggcag gcatcatata agatcttcaa aatggaaaag 720 gggaaagtaa ttaaatcagt cgaattggat gctcctaatt accactatga ggaatgctcc 780 tgttatcctg atgccggcga aatcacatgt gtgtgcagag ataattggca tggctcaaat 840 cggccatggg tgtccttcaa tcaaaatctg gagtatcaaa taggatatat atgcagtgga 900 gtttcggag acaatccacg ccccaatgat ggaacaggta gttgcggtcc ggtgtcccat 960 aatggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020 agaaccaaaa gccctaattc caggagcggc tttgagatga tttgggatcc aaatgggtgg 1080 agtgaaacgg acagtagctt ctcggtgaaa caagatatag tagcaataac tgattggtca 1140 ggatatagcg ggagttttgt ccagcatcca gaactgacaggtttagattg cataagacct 1200 tgtttctggg ttgaattaat cagagggcgg cccaaagaga gtacagtttg gactagtggg 1260 agcagcatat ccttttgtgg tgtaaatagt gacactgtga gttggtcttg gccagacggt 1320 gctgagttgc cattcagcat tgacaagtag 1350

Claims (18)

서열번호 7로 표시되는 유전자와 상동성 95% 이상인 유전자로서, 서열번호 8, 서열번호 9, 서열번호 10 및 서열번호 11로 구성된 군에서 선택되는 어느 하나의 유전자; 서열번호 12로 표시되는 유전자와 상동성 95% 이상인 유전자로서, 서열번호 13, 서열번호 14, 서열번호 15 및 서열번호 16으로 구성된 군에서 선택되는 어느 하나의 유전자; 및 서열번호 1 내지 6으로 표시되는 6개의 내부 유전자를 포함하는 인플루엔자 재배열 바이러스를 포함하는 조류 인플루엔자 바이러스 감염 예방 또는 치료용 백신.
A gene having a homology of 95% or more with the gene represented by SEQ ID NO: 7, wherein the gene is any one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; A gene having a homology of 95% or more with the gene of SEQ ID NO: 12, wherein the gene is any one selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; And an influenza rearranged virus comprising the six internal genes represented by SEQ ID NOS: 1 to 6. The present invention also relates to a vaccine for preventing or treating avian influenza virus infection.
삭제delete 삭제delete 삭제delete 제 1항에 있어서,
상기 인플루엔자 재배열 바이러스는 조류인플루엔자 재배열 바이러스인 것을 특징으로 하는 백신.
The method according to claim 1,
Wherein said influenza rearranged virus is avian influenza rearranged virus.
제 1항에 있어서,
상기 백신은 저온 적응된 형질을 나타내는 것을 특징으로 하는 백신.
The method according to claim 1,
Wherein the vaccine exhibits a cold adapted trait.
제 1항에 있어서,
상기 백신은 약독화된 형질을 나타내는 것을 특징으로 하는 백신.
The method according to claim 1,
Wherein the vaccine exhibits an attenuated trait.
서열번호 7로 표시되는 유전자와 상동성 95% 이상인 유전자로서, 서열번호 8, 서열번호 9, 서열번호 10 및 서열번호 11로 구성된 군에서 선택되는 어느 하나의 유전자; 서열번호 12로 표시되는 유전자와 상동성 95% 이상인 유전자로서, 서열번호 13, 서열번호 14, 서열번호 15 및 서열번호 16으로 구성된 군에서 선택되는 어느 하나의 유전자; 및 저온 적응된 약독화된 x-31 ca 균주의 서열번호 1 내지 6으로 표시되는 6개의 내부 유전자를 재배열시켜서 인플루엔자 재배열 바이러스를 제조하는 방법.
A gene having a homology of 95% or more with the gene represented by SEQ ID NO: 7, wherein the gene is any one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; A gene having a homology of 95% or more with the gene of SEQ ID NO: 12, wherein the gene is any one selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; And rearranging the six internal genes represented by SEQ ID NOS: 1 to 6 of the attenuated x-31 ca strain at low temperature to produce influenza rearranged virus.
삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제 1항에 있어서,
상기 백신은 이종 조류인플루엔자 바이러스에 교차 면역 반응을 형성하는 것을 특징으로 하는 백신.
The method according to claim 1,
Wherein said vaccine forms a cross-immune response to a heterologous avian influenza virus.
제 1항에 있어서,
상기 백신은 경구 또는 비강내 투여용으로 제형화된 것을 특징으로 하는 백신. The method according to claim 1,
Wherein said vaccine is formulated for oral or intranasal administration.

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