KR101582163B1 - 약독화된 뎅기 혈청형 2 균주 - Google Patents
약독화된 뎅기 혈청형 2 균주 Download PDFInfo
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- KR101582163B1 KR101582163B1 KR1020147001614A KR20147001614A KR101582163B1 KR 101582163 B1 KR101582163 B1 KR 101582163B1 KR 1020147001614 A KR1020147001614 A KR 1020147001614A KR 20147001614 A KR20147001614 A KR 20147001614A KR 101582163 B1 KR101582163 B1 KR 101582163B1
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Abstract
본 발명은 PDK 세포 및 Vero 세포에서 계대 배양함으로써, 야생형 뎅기-2 균주 16681로부터 파생된 약독화된 신규한 VDV2(Vero-Derived Dengue serotype 2 virus) 생균주에 관한 것이다. 또한, 본 발명은 VDV2 균주를 포함하는 백신 조성물에 관한 것이다.
Description
본 발명은 PDK 세포에서 계대 배양하고 Vero 세포에서 위생처리한, 야생형 뎅기-2 균주 16681 유래의 약독화된 신규한 VDV2(Vero-Derived Dengue serotype 2 virus) 생균주에 관한 것이다. 또한, 본 발명은 VDV2 균주를 포함하는 백신 조성물에 관한 것이다.
뎅기병은, 플라비바이러스(flavivirus) 속(Gubler, 1988)에 속하는, 항원성으로 구별되지만 매우 밀접하게 관련되어 있는 4가지 타입의 바이러스 혈청형에 의해 야기된다(Gubler, 1988; Kautner et al., 1997; Rigau-Perez et al., 1998; Vaughn et al., 1997). 뎅기 바이러스 혈청형에 감염되면, 비특이적인 바이러스성 증후군에서부터 치명적인 중증 출혈성 질환에 이르는 넓은 범위의 임상 질환이 발생할 수 있다. 뎅기열(DF)의 잠복기는 모기에 물린 후 평균적으로 4일(3-14일)이다. DF는 2상성 열(biphasic fever), 두통, 다양한 신체 부위의 통증, 쇠약, 발진, 림프절 병증(lymphadenopathy) 및 백혈구 감소증을 특징으로 한다(Kautner et al., 1997; Rigau-Perez et al., 1998). 바이러스 혈증의 기간(viremic period)은 열성 질환과 동일하다(Vaughn et al., 1997). DF로부터 완전히 회복되는데에는 통상 7 내지 10일이 소요되지만, 무력증이 장기간 계속되는 것이 일반적이다. 백혈구와 혈소판의 수 감소도 흔하게 나타난다.
출혈성 뎅기열(Dengue haemorrhagic fever, DHF)은 항상성의 이상과, 종종 심각한 내출혈을 동반한 저혈량증 및 저혈압(뎅기 쇼크 증후군, DSS)을 유도할 수 있는 혈관 투과성 증가를 특징으로 하는 중증 열성 질환이다. DHF의 치사율은 무치료시 10%로 높으며, 치료를 받는 경우에는 대부분 1% 미만일 수 있다(WHO Technical Guide, 1986).
뎅기 감염에 대한 일반적인 실험적 진단은 바이러스 분리 및/또는 뎅기 바이러스 특이적인 항체의 검출을 근거로 한다.
뎅기병은 말라리아 다음으로 두번째로 가장 중요한 열대성 감염 질환으로, 전세계 인구의 1/2 이상(25억)이 유행성 확산 위험성이 있는 지역에 살고 있다. 매년 뎅기병의 사례는 50,000,000 내지 100,000,000 건으로 추산되고 있으며, 이중 500,000건은 DHF로 입원하며, 25,000건은 사망한다. 뎅기는 아시아, 태평양, 아프리카, 라틴 아메리카 및 카리브해에 만연되어 있다. 100개국 이상의 열대 국가에는 풍토성의 뎅기 바이러스 감염증이 있으며, 이들 국가중 60개국 이상에서 DHF가 보고되었다(Gubler, 2002; Monath, 1994). 다음과 같은 특징적인 다수개의 인자들이 뎅기 감염증에 관여하는 것으로 보인다: 인구 증식(population growth), 특히 빈곤과 관련하여 무계획 및 무통제성 도시화, 비행기 이용 증가, 효과적인 모기 방제 부족, 및 위생 및 공공 건강 기반 시설의 퇴보(Gubler, 2002). 여행자 및 국외 거주자들 사이에서 뎅기에 대한 인식이 증가하고 있다(Shirtcliffe et al., 1998). 뎅기-유행성 열대지역에서 유행하는 기간동안에 미국 부대에서 발생되는 열병의 주된 원인은 뎅기인 것으로 입증되었다(DeFraites et al., 1994).
뎅기열 바이러스는 낮에 활동하는 인간 흡혈에 선호적인 가주성 모기인 이집트 숲 모기(Aedes aegypti) 및 인간과 연관된 사이클로 유지된다. 인간 감염은, 먼저 감염된 이집트 숲 모기가 흡혈하는 동안에 바이러스가 주입되어 개시된다. 타액의 바이러스는 혈관외 조직에 주로 머무른다. 주입된 후 감염되는 일차 세포 서브세트는 수지상 세포이며, 이후 유출성 림프절로 이동한다(Wu et al., 2000). 바이러스는 피부와 유출성 림프절에서 일차 복제된 후, 일반적으로 3 내지 5일간 급성 발열기 동안에 혈액에 출현한다.
단핵구 및 대식세포는 뎅기 바이러스의 일차 표적물 중에서도 수지상 세포와 함께 한다. 상동 기관의 재감염에 대한 방어는 완전하며 아마 평생 지속되지만, 뎅기 타입들 간의 교차 방어는 12주 미만으로 지속된다(Sabin, 1952). 그 결과, 개체는 다른 혈청형으로 이차 감염을 겪을 수 있다. 이차 뎅기 감염은 중증 뎅기병으로 발병할 수 있는 이론적인 위험 인자이다. 그러나, DHF는, 연루된 바이러스 균주 뿐만 아니라 환자의 연령, 면역 상태 및 유전적 소인을 포함하는, 다인자성 질환이다. 다음의 2가지 인자가 DHF 발생에 주된 역할을 한다: 높은 바이러스 혈증을 보이는 바이러스의 빠른 복제(질병의 심각성은 바이러스 혈증 정도와 관련있음)(Vaughn et al., 2000) 및 염증 매개체를 높은 수준으로 방출하는 중요한 염증 반응(Rothman and Ennis, 1999).
뎅기병에 대한 특이 치료제는 없는 실정이다. DF 처치는 침상 휴식, 해열제와 진통제에 의한 열 및 통증 통제, 및 적절한 수액 섭취로 이루어진다. DHF의 치료에는 체액 손실 보정, 응혈 인자의 대체 및 헤파린 주입이 요구된다.
현재 예방 조치들은 병원균 매개 동물의 방제와 개인적인 보호 조치에 의지하고 있지만, 이는 수행하기 어려우며 비용이 많이 든다. 현재, 승인된 뎅기 백신은 없다. 4가지 뎅기 혈청형이 전세계적으로 순환되고 있으며 DHF에 관여되는 것으로 보고되고 있기 때문에, 백신 접종은 이상적으로는 4가지 뎅기 바이러스 혈청형 모두에 대한 방어를 부여할 수 있어야 한다.
약독화된 생백신(Live attenuated vaccine, LAV)은 선천적인 면역성을 재생시키는 것으로, 뎅기와 동일한 종에 속하는 일부 바이러스를 포함하여, 수많은 질환에 대한 백신 개발에 사용되고 있다(예, 상업적으로 구입가능한 플라비바이러스 약독화된 생백신으로는 황열 및 일본 뇌염 백신이 있음). 약독화된 생바이러스 백신의 이점은 그것의 복제 능력과 체액성 및 세포성 면역 반응 둘다를 유도할 수 있는 능력이 있다는 것이다. 아울러, 전체 비리온 백신에 의해 유도된 바이러스의 여러가지 성분(구조 및 비구조 단백질)들에 대한 면역 반응은 자연적인 감염에 의해 유발되는 면역 반응을 재현한다는 것이다.
뎅기 백신 계획은 태국에 소재한 마히돌 대학의 과학기술 개발 기구인 백신 개발 센터에서 시작되었다. 약독화된 생백신에 대한 후보 백신이, PDK(primary dog kidney) 세포에서 뎅기 혈청형 1(균주 16007, 계대 13 = LAV1), 혈청형 2(균주 16681, 계대 53) 및 혈청형 4(균주 1036, 계대 48) 바이러스에 대해, 그리고, PGMK(Primary Green Monkey Kidney) 세포(계대 30) 및 FRhL(Fetal Rhesus Lung) 세포(계대 3)에서 혈청형 3(균주 16562)에 대해, 실험실 규모로 성공적으로 개발되었다. 이들 백신들은 태국의 지원자를 대상으로 1가(단일 혈청형), 2가(2종의 혈청형), 3가(3종의 혈청형) 및 4가(4종 모두의 혈청형) 백신으로서 테스트 되었다. 이들 백신들은 어린이 및 성인에서 안전하며 면역원성이 있는 것으로 확인되었다(Gubler, 1997). 이들 LAV 1-4 균주들은 마히돌 대학의 EP 1159968에 언급되어 있으며, 그 이전에 CNCM(CNCM I-2480; CNCM I-2481; CNCM I-2482 및 CNCM I-2483 각각)에 기탁되었다.
Den-2 균주 16681은 1964년 방콕에서 DFH(Dengue Hemorrhagic Fever) 환자의 혈청에서 회수되었다(Halstead et al., 1970). 바이러스 혈증을 보이는 최초 혈청은 BSC-1 세포(African Green Monkey kidney cell)에서 4번 계대 배양되었고, LLC-MK2 세포(Rhesus Monkey kidney cell)에서 5번 계대 배양되었다. 1977년에, 바이러스는 감수성 원숭이(Macaca Mulatta)에서 1회 생체내 계대 증폭되었고, 다시 LLC-MK2 세포에서 계대 배양되었다. 1980년에는 모기(Toxorhynchites amboinensis)에서 2번의 계대 증폭이 수행되었다. 바이러스 약독화는 PDK 세포(primary dog kidney cell)에서 32 ℃로 계대 배양에 의해 수행되었다. 균주의 약독화는 시험관내 및 생체내 마커 수종에 의해 체크되었다. 50회의 계대 배양시 이러한 약독화 기준을 모두 만족시켰으며, 53회 계대 배양물이 백신 생산용 마스터 종균(1982)으로서 선정되었다. DEN-2 PDK53 백신 후보물질은 인간에서 평가되었으며, 면역성이 높으며 원하지 않는 임상적인 신호나 증상은 없는 것으로 확인되었다.
약독화된 뎅기 2 바이러스 생균주(LAV2)의 전체 서열은 2001년에 R. Kinney 등(CDC, Fort Collins)에 의해 확립되었다. 모균주 DEN-2 16681(서열번호 3)과 LAV2(서열번호 38) 균주들 간의 서열 차이는 표 1에 나타낸다. 이에, 야생형 바이러스 균주인 16681과 LAV2 균주의 유전자를 비교한 결과, LAV2 약독화와 연결지을 수 있는 9개의 점 돌연변이 세트가 확인되었다.
DEN-2 16681 및 DEN-2 16681/PDK53(LAV2)의 서열 차이
해당 코돈을 변형시키는 뉴클레오티드 변화는 볼드체로 나타낸다.
1983년에 최초로 확립된 LAV2 균주는, 또한 가능성 있는 백신 후보로 신속하게 확정되었다(Bhamarapravati and Yoksan, 1997).
그러나, 그 당시에 포유류 배양물을 통한 해면상 뇌염(Spongiform Encephalitis)의 인간 전파가 위험한 것으로 인지되지 않았으며, 바이러스는 통상 PDK(Primary Dog Kidney) 세포에서 유지되었다. 더욱이, 이 LAV2 균주는 혼성(heterogeneous) 개체이다. 이러한 혼성(heterogeneity)은 조성물에 존재하는 하나의 균주의 시험관내 또는 생체내 선별 가능성이 있어, 추가적인 위험이 있음을 의미한다.
이러한 우려가 증가되어, 출원인은 이러한 위험으로부터 벗어나기 위한 위생 공정을 확립하기로 결심하였다. LAV2 백신 균주를 PDK에서 Vero 세포로 먼저 이동시킨 다음 LAV2의 정제된 게놈 RNA를 Vero 세포에 형질감염시키고, 2번의 연속적인 바이러스의 플러그 정제 단계를 실시하여, 출원인은 신규한 Vero-유래 혈청형 2 바이러스(VDV2)를 만들었다.
Vero 세포로 전이되어 생물학적으로 클로닝됨으로써 파생된 신규한 VDV2 균주는, 서열, 동질적인 플라그 크기 및 온도 민감성이 LAV2 균주와 다르지만, 예컨대 감퇴 스팟(attenuation spot), 작은 플라그 표현형, 고온에서의 증식 제한과 같은 LAV2의 일부 표현형 및 유전자형 특징들이 보존되어 있으며, LAV2 균주의 면역원 특징들도 보존되어 있다는 점에서 중요하다. 이러한 특징들로 인하여, 상기 신규 균주는 예방 측면에서 인간을 면역화시키는 유용한 백신 후보물이 될 수 있다.
본 발명은 PDK 세포 및 Vero 세포에서 계대 배양함으로써, 야생형 뎅기-2 균주 16681로부터 파생된 약독화된 신규한 VDV2(Vero-Derived Dengue serotype 2 virus) 생균주 및 VDV2 균주를 포함하는 백신 조성물을 제공한다.
정의
"뎅기 바이러스"는 플라비리다에(flaviridae) 과의 플라비바이러스 속에 속하는, (+) 단일가닥 RNA이다. 뎅기 혈청형2(DEN-2) 균주 16681의 경우, 전체 서열은 10723개의 뉴클레오티드(서열번호 3)로 이루어져 있다. RNA 게놈에는 5'말단에 타입 1 캡(cap)이 있으며, 3'-말단 폴리(A)-테일(tail)은 없다. 유전자 구성은 5'-비코딩부(NCR), 구조 단백질(캡시드(C), 프리멤브레인/멤브레인(prM/M), 엔벨로프(E)), 비구조 단백질(NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5), 및 3' NCR이다. 바이러스 RNA 게놈은 C 단백질과 조합되어 뉴클레오캡시드(20면체 대칭)를 형성하고 있다. 다른 플라비바이러스와 같이, DEN 바이러스 게놈은 하나의 폴리단백질로 번역되는, 연속적인 오픈 리딩 프래임(ORF)을 코딩하고 있다.
뎅기-2의 병원성(질병을 유발하는) 균주를 수회 계대 배양하면, "살아있는 약독화"된, 즉 감염성은 있지만 질병은 유발하지 못하는, 변형된 바이러스 분리물이 생긴다. 이렇게 변형된 바이러스는 일반적으로 원숭이에서 테스트하여, 이들의 약독성을 평가한다. 그러나, 인간은 임상 질환의 증상을 나타내는 유일한 영장류이다. 테스트한 인간 대부분에서 (즉, 긍정적인 효과/위험 비로 표시되는, 규제하기 위한 측면에서 허용가능한) 중간 내지 낮은 효과를 유발하거나, 또는 부차적인 효과(즉, 전신 현상 및/또는 생물학적인 비정상성 및/또는 국부 반응)를 유발하지 않지만, 여전히 감염성을 보이며 면역 반응을 유도하는 바이러스를 "약독화된 생" 바이러스라고 한다.
용어 "LAV"는 약독화된 뎅기 생바이러스 균주를 나타낸다. 본 발명에서, "LAV"는 PDK 세포에서의 계대 배양에 의해 뎅기 혈청형 2(DEN-2) 균주 16681로부터 최초로 파생된 약독화된 생균주이다. 예컨대, "LAV2/PDK53"은 PDK 세포에서 균주 16681을 53회 계대 배양한 후 확립한 약독화된 균주이다(또한, DEN-2 16681/PDK53이라 명명됨). "LAV2/PDK50"은 PDK 세포에서 균주 16681을 50회 계대 배양한 후 확립한 약독화된 균주이다(DEN-2 16681/PDK50). LAV2/PDK53의 뉴클레오티드 서열은 서열번호 38에 나타낸다.
"VDV2"는 본 발명에 개시된 위생 처리 공정에 의해 수득가능한 LAV를 의미한다. 따라서, VDV2는 영장류, 특히 인간에서 중화 항체를 포함하여, 특이적인 체액성 면역 반응을 유도할 수 있는, 생물학적 클론(동일한) Vero-순응된 뎅기 혈청형2 바이러스이다. 본 발명의 VDV2 균주는 개시된 VDV2 서열로부터 출발하여 직접 쉽게 재구축할 수 있다. 특이적인 체액성 면역 반응의 유도는 ELISA 분석에 의해 용이하게 측정할 수 있다. 백신을 접종받은 개체의 혈청내 중화 항체의 존재는 섹션 4.1.1.2.2.에 언급된 바와 같이 플라그 감소 중화 테스트(plaque reduction neutralization test)로 평가한다. 그 결과 결정된 중화 항체의 역가가 1:10 보다 높거나 또는 1:10이면, 혈청은 중화 항체의 존재에 대해 양성인 것으로 간주된다.
용어 "돌연변이"는 유전적 물질, 예컨대 DNA, RNA, cDNA 또는 모든 과정, 기작의 검출가능한 모든 변화 또는 그러한 변화의 결과를 의미하다. 돌연변이는 한개 이상의 뉴클레오티드의 치환을 포함한다. 본 발명에서, 뎅기-2 바이러스 게놈 서열 또는 폴리단백질에서 확인된 돌연변이는 Dunnen 및 Antonarakis(2000)의 명명법에 준하여 명명된다. Dunnen 및 Antonarakis에 의해 정의된 바와 같이, 핵산 수준에서 치환은 ">"로 표시되며, 예로, "31A>G"는 기준 서열의 31번 뉴클레오티드가 A에서 G로 변경됨을 나타낸다.
단백질에서의 변이는 돌연변이의 결과를 나타내며, 하기와 같이 기재된다. 정지 코돈은 X로 명기된다(예, R97X는 Agr96의 종결 코돈으로의 변경을 나타냄). 아미노산 치환은 예컨대, 9번 Ser이 Gly으로 치환됨을 의미하는 "S9G"로 표시된다.
ero
유래 혈청형 2의
뎅기
바이러스(
VDV2
)
기존에 개발된 뎅기-2 백신 후보체 LAV2의 조성물을 위생처리 공정에 의해 개량하였다.
본원에 개시된 Vero-유래 혈청형 2의 뎅기 바이러스(VDV2)는 PDK 세포에서의 연속적인 계대 배양에 의해 약독화된 DEN-2 16681 바이러스를 이용한다. VDV2는 약독화된 DEN-2 생바이러스의 전체 게놈 서열을 포함하고 있으며, 인간을 대상으로 테스트한 LAV2 원균주와 같이 약독화로 연결되는 동일한 스팟을 가지고 있다.
LAV2 백신 후보체의 위생 처리는 단백질 제거 단계 및 정제된 바이러스 게놈 물질만을 Vero 세포에 도입하는 단계에 의해 수행되었다. 보다 상세하게는, 균주의 위생 처리는 정제 단계 및 바이러스 RNA의 Vero 세포로의 형질전환시키는 단계로 수행되었다. 방법은 하기 단계를 포함한다:
a) 플라그-정제한 LVA2 균주, 예컨대 DEN-2 16681/PDK50 바이러스에서 바이러스 RNA를 추출 및 정제하는 단계;
b) 정제한 RNA를 양이온성 지질과 효과적으로 조합시키는 단계;
c) Vero 세포, 특히 Vero 세포 LS10에 형질감염시키는 단계;
d) 신규-합성된 바이러스를 회수하는 단계; 및
e) 플라그 정제에 의해 VDV 균주를 정제하고, 선택적으로 숙주 세포, 특히 Vero 세포에서 이를 증폭시키는 단계.
Vero 세포 기법은 여러가지 상업적인 제품(주사용 및 경구 폴리오 백신, 광견병 백신)에 사용되는 잘 알려져 있는 기법이다. 본 발명에서 이롭게는 외래 물질(adventitious agent)이 존재할 어떠한 위험도 없음을 보장하기 위해, 검증된(qualified) Vero 세포를 사용하였다. 본 발명에서, "검증된 Vero 세포"는, 배양 조건이 알려져 있는 세포 또는 세포주를 의미하며, 상기 세포는 임의 외래 물질이 없는 것이다. 이것으로는, 예컨대 Sanofi Pasteur의 Vero 세포 LS10이 있다.
그에 따라 분리된 VDV 균주는 동결 조성물 또는 동결건조 산물 형태 중 어느 한가지 형태로 관행에 따라 보관된다. 이를 위해, VDV는 희석제, 통상적으로 당 알코올 및 안정화제와 같은 동결보존성 화합물을 포함하는 완충화된 수용액과 혼합될 수 있다. 동결 또는 동결건조하기 전 pH는, 실온에서 pH 측정기로 측정시, 6 내지 9, 예컨대 pH 7.5 + 0.2와 같이 약 7로 설정하는 것이 좋다. 사용하기 전에, 동결건조된 산물은, 멸균 NaCl 4‰ 용액과 같은 약제학적인 희석제 또는 부형제와 혼합되어, 면역원성 액체 조성물 또는 백신으로 재구성된다.
LAV2 백신 균주의 NS3-250 위치의 Glu 변이체를 형질감염 및 클로닝하면서 선별하였으며, 또한 LAV2 백신의 약독화에 대한 기준으로서 동정된 5'NC-57 및 NS1-53 역시 VDV2 서열에서도 모두 보존되어 있었다.
형질감염한 후 회수된 바이러스의 약독화-특이 부위를 서열분석하였으나, 서열번호 38과 비교하여 어떠한 돌연변이도 확인되지 않았다. 생물학적으로 클로닝된 VDV2 바이러스는, 특히 약독화 표현형의 보존으로부터 유출할 수 있는 것처럼, 이의 LAV2 모균주와 상동한 플라그 표현형과 상당한 유전적 안정성을 갖는다.
VDV2 균주(11회 계대)를 서열 분석하였고, 혈청형 2 뎅기의 약독화된 생바이러스(LAV2) 균주의 서열(서열번호 38)과 비교하였다. M 및 Env 구조 펩티드와 또한 비구조 펩티드 NS3 및 NS5에 위치된 6개의 아미노산 치환을 촉발시키는 10개의 뉴클레오티드 차이가 확인되었다. 이러한 차이는 LAV2의 약독화 위치 중 어디에도 있지 않았다.
LAV2/PDK53 및 VDV2의 11회 계대 배양 균주 간의 서열 비교
| 뉴클레오티드 | 아미노산 | ||
| 위치 | LAV2 VDV2 | 부위 위치 | LAV2 VDV2 |
| 736 1619 1638 2520 4723 5062 9191 9222 10063 10507 |
G C G A A G G A T A G C G A A G T A A G |
M 9 E 228 E 234 NS1 33 NS3 69 NS3 181 NS5 541 NS5 551 NS5 832 3'nc - |
G R G E K K K K S T D H R K E E S T - - |
회색 음영: 구조 단백질에서의 차이; 볼드체: 비구조 단백질에서의 차이
따라서, 본 발명은 PDK 세포에서 연속 계대 배양하고, 이후 Vero 세포에서 위생처리함으로써, 약독화된 야생형 DEN-2 16681 바이러스로부터 수득된 약독화된 뎅기-2 바이러스 생균주를 제공한다. 특히, 본 발명의 약독화된 균주는 야생형 DEN-2 16681 및 LAV2/PDK53 균주의 뉴클레오티드 서열 또는 폴리단백질 서열을 기준으로, 확인된 하나 이상의 서열 돌연변이(무-침묵 및 선택적으로 침묵)를 포함한다.
따라서, 본 발명은 LAV2/PDK53 균주(서열번호 38)의 서열을 포함하는, 또는 상기 서열로 구성된, 분리된 약독화된 뎅기-2 바이러스 생균주에 관한 것으로, 상기 서열에서 736, 1619, 4723, 5062, 9191, 10063 및 10507번, 그리고 선택적으로 1638, 2520, 9222 및 10361번의, 뉴클레오티드 중 한 개 이상은 돌연변이되어 있으며, 단, 57, 524, 2055, 2579, 4018, 5547, 6599 및 8571번의 뉴클레오티드는 돌연변이 되어 있지 않다. 바람직하기로는, 돌연변이는 치환이다. 바람직하기로는, 736번의 뉴클레오티드는 C이고, 1619번의 뉴클레오티드는 A이고, 4723번의 뉴클레오티드는 A이고, 5062번의 뉴클레오티드는 A이고, 9191번의 뉴클레오티드는 A이고, 10063번의 뉴클레오티드는 A이고, 10507번의 뉴클레오티드는 G이다.
5270번의 뉴클레오티드는 A 또는 T이며, 바람직하기로는, A이다.
더 바람직하기로는, 본 발명에 따라 분리된 균주는 돌연변이 736 G>C, 1619 G>A, 4723 T>A, 5062 G>C, 9191 G>A, 10063 T>A 및 10507 A>G와, 선택적으로 1638 A>G, 2520 G>A 및/또는 9222 A>G를 포함하는, 서열번호 38의 서열을 포함한다.
이에, 본 발명에 따른 약독화된 뎅기-2 바이러스 생균주는, 57 C>T, 524 A>T, 736 G>C, 1619 G>A, 2055 C>T, 2579 G>A, 4018 C>T, 4723 T>A, 5062 G>C, 5547 T>C, 6599 G>C, 8571 C>T, 9191 G>A, 10063 T>A 및 10507 A>G 중 한가지 이상의 돌연변이를 포함하는, 야생형 뎅기-2 균주 16681(서열번호 3)의 서열을 포함하거나, 또는 상기 서열로 구성될 수 있다. 바람직하기로는, 본 발명에 따른 약독화된 생균주는 야생형 균주 16681의 뉴클레오티드 서열(서열번호 3)을 기준으로 1638 A>G, 2520 G>A 및/또는 9222 A>G 돌연변이를 더 포함한다.
본 발명에 따른 약독화된 뎅기-2 바이러스 생균주는, 전술한 바와 같이, 736, 1619, 4723, 5062, 9191, 10063 및 10507번이 돌연변이된 서열번호 3의 서열을 포함하고 있으며, 해당 코돈 위치에서 코딩하고 있는 아미노산을 변이시키지 않고서 그 코돈 위치에 하나 이상의 뉴클레오티드 치환을 더 포함하고 있는 변이체 균주이다.
이롭게는, 본 발명에 따른 약독화된 뎅기-2 바이러스 생균주는 서열번호 1에서 한정된 수의 돌연변이, 예컨대 5개 이하, 바람직하기로는 2개 이하의 돌연변이가 있는, 서열을 포함한다.
바람직하기로는, 본 발명에 따른 뎅기-2 바이러스의 게놈 서열은 서열번호 1의 뉴클레오티드 서열로 구성된다.
또한, 본 발명은 세포, 특히 Vero 세포에서의 추가적인 계대 배양에 의해 서열번호 1의 서열을 가진 VDV2 균주로부터 파생될 수 있는, 약독화된 뎅기-2 생균주에 관한 것이다.
또한, 본 발명은 서열번호 1의 DNA 서열 또는 이의 등가 RNA 서열을 포함하거나, 또는 서열로 구성되는 분리된 핵산에 관한 것이다.
"핵산 분자"는, 단일가닥 형태 또는 이중가닥 나선형의, 리보뉴클레오시드(아데노신, 구아노신, 우리딘 또는 시티딘; "RNA 분자") 또한 데옥시리보뉴클레오시드(데옥시아데노신, 데옥시구아노신, 데옥시티미딘 또는 데옥시시티딘; "DNA 분자")의 포스페이트 에스테르 폴리머형, 또는 포스포로티오에이트 및 티오에스테르와 같은 이의 모든 포스포에스테르 유사체이다.
본원에서, 서열번호 1의 RNA 서열 "등가체"는 서열번호 1에서 데옥시티미딘이 우리딘으로 치환된 것을 의미한다. 서열번호 1은 VDV2 cDNA 서열을 구성하므로, 따라서 이의 RNA 등가 서열은 VDV2의 (+) RNA 가닥에 해당된다.
본 발명은 또한 서열번호 2의 서열을 포함하는 폴리단백질 및 이의 단편에 관한 것이다. 서열번호 2는 서열번호 1에 의해 엔코딩된 폴리단백질 서열이다.
기준 단백질의 "단편"은 기준 단백질의 보존적인 아미노산 체인을 포함하는 서열의 폴리펩티드이다. 단편의 아미노산 길이는 8개 이상, 12개 이상 또는 20개 이상일 수 있다.
서열번호 2의 폴리단백질의 단편은 적어도 M 단백질의 9번(서열번호 2의 214번)에 아르기닌, 및/또는 E 단백질의 228번(서열번호 2의 508번)에 글루탐산, 및/또는 NS3 단백질의 69번(서열번호 2의 1543번)에 트레오닌, NS3 단백질의 181번(서열번호 2의 1656번)에 히스티딘, 및/또는 NS5 단백질의 541번(서열번호 2의 1725번)에 라이신, 및/또는 NS5 단백질의 832번(서열번호 2의 3032번)에 트레오닌을 포함한다.
예로, 서열번호 1에 의해 엔코딩되는 폴리단백질의 단편은 M 단백질, E 단백질, NS3 단백질 및/또는 NS5 단백질이거나 또는 이를 포함한다.
면역성 및 백신 조성물
또한, 본 발명은 본 발명에 따른 VDV2 균주를 포함하는, 백신으로서 사용하기에 적합한, 면역성 조성물에 관한 것이다.
본 발명에 따른 면역성 조성물은 중화 항체를 포함하는 뎅기 바이러스에 대해 특이적인 체액성 면역 반응을 도출한다.
바람직하기로는, 면역성 조성물은 백신이다.
예로, 면역성 조성물은 일가 조성물이며, 즉, 이 조성물은 뎅기-2 바이러스에 대해서만 특이적인 면역 반응을 나타내거나/나타내고, 방어를 부여한다.
다른 예로, 본 발명은 다가 뎅기 면역성 조성물에 관한 것이다. 이러한 다가 면역성 조성물 또는 백신은 개별적인 일가 뎅기 백신들을 조합함으로써 수득할 수도 있다. 면역성 조성물 또는 백신 조성물은 다른 혈청형의 약독화된 뎅기 생바이러스 하나 이상을 추가적으로 포함할 수 있다. 특히, 면역성 조성물 또는 백신 조성물은, 본 발명에 따른 VDV2를, 혈청형 1, 3 및 4로 이루어진 군으로부터 선택된 한가지 이상의 약독화된 뎅기 생바이러스와 조합하여, 포함할 수 있다.
바람직하기로는, 면역성 조성물 또는 백신 조성물은, 4가 뎅기 백신 조성물, 즉 본 발명에 따른 VDV2를 약독화된 뎅기-1 바이러스 생균주, 약독화된 뎅기-3 바이러스 생균주 및 약독화된 뎅기-4 바이러스 생균주와 조합하여 포함하는 백신 조성물일 수 있다.
약독화된 뎅기-1, 뎅기-3 및 뎅기-4 바이러스 생균주는 이미 개시되어 있다. 뎅기 혈청형1(16007 균주, 13회 계대 배양; LAV1) 및 혈청형 4(1036 균주, 48회 계대 배양, LAV4) 바이러스를 PDK(Primary Dog Kidney) 세포에서, 혈청형 3(16562 균주)을 PGMK(Primary Green Monkey Kidney) 세포(30회 계대 배양) 및 FRhL(Fetal Rhesus Lung) 세포(3회 계대 배양)(LAV3)에서, 계대 배양하여 얻어진, 마히돌 대학에서 개발한 약독화된 생 백신을 참조할 수 있다. LAV1(서열번호 40), LAV3(서열번호 41) 및 LAV4(서열번호 42)의 뉴클레오티드 서열들은 첨부한 서열목록에 나타나있다.
이롭게는, 약독화된 뎅기-1 생균주는, 마히돌 대학에서 개발한 LAV1 균주를 Vero 세포에서의 위생 처리 공정에 의해 수득한 VDV1 균주일 수 있다. 특히, 약독화된 뎅기-1 생균주(VDV1)는 서열번호 39의 서열을 포함할 수 있으며, 이롭게는 상기 서열로 구성될 수 있다.
백신을 포함한 면역성 조성물은 액체 용액, 현탁액 또는 유제일 수 있는 주사제로서 제조할 수 있다. 활성 면역성 성분을 이와 혼용가능한 약제학적으로 허용가능한 부형제와 혼합할 수 있다.
본 발명에 따른 면역성 조성물 또는 백신은 당업자에게 공지된 모든 통상적인 방법을 이용하여 제조할 수 있다. 통상적으로, 본 발명에 따른 항원을 물 또는 포스페이트 인산화된 염수 용액, 습윤제, 충진제, 유화제, 안정화제와 같은 약제학적으로 허용가능한 희석제 또는 부형제와 혼합한다. 부형제 또는 희석제는 투여 방법과 경로, 그리고 약학 실무에 따라 선택되는 약학 형태에 따라, 선택될 것이다. 적합한 부형제 또는 희석제, 그리고 약학 제형의 요건은 약학 분야의 참고 도서인 Remington's Pharmaceutical Sciences에 기재되어 있다.
바람직하기로는, 면역성 조성물 또는 백신은 예컨대 pH(실온에서 pH 측정기로 측정하는) 6 내지 9를 유지하도록 완충화된 수용액을 포함하는, 주사 조성물이다.
본 발명에 따른 조성물은 보강제, 즉 VDV2 균주에 의해 도출되는 면역 반응을 개선 또는 강화하는 물질을 더 포함할 수 있다. 인간 백신에 일반적으로 사용되는 약제학적으로 허용가능한 보강제 또는 보강제 혼합물을 상기한 목적으로 사용할 수도 있다.
본 발명에 따른 면역성 조성물 또는 백신은, 본 발명에서 비경구(예, 진피내, 피하, 근육내) 경로와 같이, 인간 백신 분야에서 일반적으로 사용되는 통상적인 경로로 투여할 수 있으며, 면역성 조성물 또는 백신은 바람직하기로는 어깨 삼각근에 피하 투여되는 주사 조성물이다.
면역화 방법
본 발명은 본 발명에 따른 면역성 조성물 또는 백신을 면역유효량으로 숙주에게 투여하는 단계를 포함하는, 면역화를 필요로 하는 숙주에 뎅기 감염을 면역화시키는 방법을 제공한다.
"면역화를 필요로 하는 숙주"는 뎅기 감염 위험이 있는 사람, 즉 뎅기 바이러스 감염이 유행중인 지역으로 여행하는 사람과 그 지역의 거주자이다.
투여 경로는 백신 분야에서 통상적으로 사용되는 모든 경로이다. 투여 경로의 선택은 선택되는 제형에 따라 결정된다. 바람직하기로는, 면역성 조성물 또는 백신은 이롭게는 삼각근에 피하 경로를 통해 투여되는 주사용 조성물이다.
면역성 조성물 또는 백신내 LAV 또는 VDV, 특히 VDV2의 양은 바이러스 플라그 형성 단위(PFU) 또는 세포 배양 감염량 50%(Cell Culture Infectious Dose 50%, CCID50)의 투여량 형태로 일반적으로 표현할 수 있으며, 통례적인 약학 기법을 이용하여 측정할 수 있다. 예로, 본 발명에 따른 조성물은 10 내지 106 CCID50, 또는 103 내지 105 CCID50의 LAV 또는 VDV를 포함하는 투약 형태로 제조할 수 있으며, 예로, 일가 조성물의 경우 VDV2 균주 4 + 0.5 log10의 CCID50을 포함할 수 있다. 조성물이 다가 조성물일 경우, 바이러스 간섭 가능성을 낮추고 그에 따라 안정된 면역 반응(즉, 조성물에 포함된 모든 혈청형에 대한 면역 반응)을 달성하기 위하여, 투여된 백신에 존재되는 여러가지 뎅기 혈청형 각각의 양은 동등하지 않을 수 있다.
"면역유효량"은 섹션 4.1.1.2.2에 개시한 바와 같이 플라그 감소 중화 테스트 평가에 따라, 백신 접종자의 혈청내 중화 항체를 포함하여 특이적인 체액성 면역 반응을 유도할 수 있는 함량이며; 그에 따라 측정된 중화 항체 역가가 1:10 이상이거나 또는 1:10일 경우 혈청에는 중화 항체가 있는 것으로 간주된다.
투여 부피는 투여 경로에 따라 변경될 수 있다. 피하 주사제의 부피는 약 0.1 ml 내지 1.0 ml, 바람직하기로는 0.5 ml일 수 있다.
조성물의 최적 투여 시간은 뎅기 바이러스에 최초 노출되기 전 약 1달 내지 3달이다. 본 발명의 백신은 뎅기 감염 위험성이 있는 성인이나 어린이를 대상으로 예방제로서 투여될 수 있다. 따라서, 표적이 되는 집단은 뎅기 바이러스에 대해 무경험자 및 경험자를 포함한다. 본 발명의 백신은 단회 투여로 투여될 수 있으며, 또는 선택적으로, 투여는 당업자가 적합하게 결정한 바에 따라 일차 접종하 다음 예컨대 2-6달 후 추가(booster) 접종으로 이루어질 수 있다.
본 발명은 하기 도면 및 실시예를 들어 보다 상세히 설명된다.
본 발명은 PDK 세포 및 Vero 세포에서 계대 배양함으로써, 야생형 뎅기-2 균주 16681로부터 파생된 약독화된 신규한 VDV2(Vero-Derived Dengue serotype 2 virus) 생균주 및 VDV2 균주를 포함하는 백신 조성물을 제공한다.
도 1은 VDV2 프리-마스터(pro-master) 종균의 역사를 종합한 것이다.
도 2는 FILLED Product(일가) "즉시 사용가능한(ready to use)" 투약제를 만드는 개발 제조 경로를 요약한 플로우 차트이다.
도 3은 VDV2 게놈 맵을 개략적으로 표시한 것이다. 상위 화살 표시는 폴리단백질의 코딩 서열이다. 하위 화살 표시는 성숙형 펩티드의 코딩 서열이다. 수직 바는 야생형 뎅기 2 균주 16681과 LAV2 균주 간의 뉴클레오티드 차이를 나타낸 것이다. 별 표시는 LAV2와 VDV2 간의 뉴클레오티드 차이를 나타낸다.
도 4는 뎅기-2 바이러스 LAV2, VDV2, 및 균주 16681을 37 ℃에서 7일간 배양한 후 플라그 크기를 분석한 것이다.
도 5는 뎅기-2 바이러스 LAV2, VDV2 및 균주 16681에 대한 플라그 크기 분포를 나타낸 그래프 분석도이다.
도 6은 플라비바이러스를 경험하지 않은 건강한 성인을 대상으로 VDV2 일가의 안전성을 평가하기 위한 실험 설계를 요약한 것이다.
도 2는 FILLED Product(일가) "즉시 사용가능한(ready to use)" 투약제를 만드는 개발 제조 경로를 요약한 플로우 차트이다.
도 3은 VDV2 게놈 맵을 개략적으로 표시한 것이다. 상위 화살 표시는 폴리단백질의 코딩 서열이다. 하위 화살 표시는 성숙형 펩티드의 코딩 서열이다. 수직 바는 야생형 뎅기 2 균주 16681과 LAV2 균주 간의 뉴클레오티드 차이를 나타낸 것이다. 별 표시는 LAV2와 VDV2 간의 뉴클레오티드 차이를 나타낸다.
도 4는 뎅기-2 바이러스 LAV2, VDV2, 및 균주 16681을 37 ℃에서 7일간 배양한 후 플라그 크기를 분석한 것이다.
도 5는 뎅기-2 바이러스 LAV2, VDV2 및 균주 16681에 대한 플라그 크기 분포를 나타낸 그래프 분석도이다.
도 6은 플라비바이러스를 경험하지 않은 건강한 성인을 대상으로 VDV2 일가의 안전성을 평가하기 위한 실험 설계를 요약한 것이다.
실시예
1: 위생 처리
1.1
바이러스
RNA
정제
RNA 정제 및 형질감염 과정은 하기와 같이 수행하였다. DEN-2/PDK50 현탁액을 0.5 ml의 물에 재현하고, ml 당 바이러스가 적어도 3 x 104 에서 최대 3 x 107 TCID50 또는 PFU로 함유되도록 희석하였다. DNA 또는 RNA 분자를 세포성 물질에서 분해시키기 위해, 윌리암 배지 0.01 ml에 희석시킨 벤조나제 1 unit을 바이러스 0.5 ml에 첨가하고, 이 용액을 교반기에서 2 시간동안 4℃에서 배양하였다. 배양 단계 후, 구아니듐 클로라이드, 용제(SDS) 및 베타-머캅토에탄올(RTL-bmercaptoethanol buffer, RNeasy Mini kit내 제공, Qiagen Ref. 74104)을 포함하는 변성 완충액 0.65 ml을 첨가하고, 페놀/클로로포름(1/1)(v/v) 1번, 클로로포름(v/v)으로 1번 단백질을 추출한 다음, 실온에서 5분간 14,000 rpm으로 원심분리하였다. 각 추출 후, 경계면의 물질(백색 침전물)을 취하지 않도록 조심스럽게 수상을 모아, 깨끗한 1 ml 에펜도르프(eppendorf) 튜브로 이동시켰다. 이후, 잔류 용매를 제거하기 위해 제조사의 권고안에 따라 RNA 용액을 QIAgen 컬럼(RNeasy minikit, QIAgen)에 적용한 다음 0.06 ml의 뉴클레오티드 결핍성 물로 용출시켰다. 바이러스 RNA의 존재는 함량을 알고 있는 바이러스로 확립한 기준 곡선을 이용하여 정량적인 RT-PCR로 TCID50/ml로 확인하였다.
1.2
정제한
RNA
의
Vero
세포 형질감염
형질감염은 전하 상호작용을 통해 RNA에 조합하여, 세포 막 융합에 의해 복합체를 세포질내로 전이시키는 양이온성 지질들의 혼합물인 리포펙타민(LF2000 Reagent, Life Technologies)을 이용하여 수행하였다. LF2000 시약의 최적량은, 리포펙타민의 양을 증가시키면서(5 내지 20 ㎕), 16 내지 24시간 전에 접종한 Vero 세포의 배양(0.3- 0.5 x 106 세포/웰, 6웰 플레이트)에 의한 예비 실험으로 결정하였다. 이후, 세포를 4 - 5 시간동안 32℃ 및 5% CO2에서 배양하고, 배지를 FCS 무첨가한 새로운 배양 배지로 교체한 다음, 32℃에서 계속하여 철야 배양하였다. 독성(둥근 세포, 굴절성 세포 또는 부유성 세포, 세포 단일층의 동질성)을 도립 현미경으로 48시간동안 규칙적으로 체크하였다. 이러한 조건에서 무독한 리포펩타민의 최대량은 10 ㎕이었으며, 이를 RNA 형질감염에 사용 수치로 선택하였다.
RNA 조제물의 1/4(약 2 x 104 log eqTCID50, qRT-PCR)을 사용하여 4세트의 형질전환을 동시에 수행하였다. 바이러스 RNA 용액 25 ㎕을 15 ㎕의 LF2000 시약(전하 상호작용을 통해 RNA와 조합하여, 세포 막 융합에 의해 복합체를 세포질내로 전이시키는 양이온성 지질들의 혼합물)을 포함하는 500 ㎕의 OptiMEM 배지 (GIBCO)로 희석하였다. 효소 tRNA 200 ng을 4개의 반응물중 2개에 담체로서 첨가하였다.
4개의 형질감염 혼합물을 10분간 실온에서 침강되게 둔 다음 컨플루언트 Vero 세포가 있는 6웰 플레이트에 첨가하고, 32℃에서 배양하였다. 4시간 후, 형질감염 혼합물은 버리고 세포를 PBS로 한번 헹구었다. 형질감염 후 배지(Williams, GIBCO) 3 ml을 첨가하여, 32℃에서 5일간 연속 배양하였다. 배양 배지는 뎅기 감염 배지(10 mM MgSO4이 첨가된 Williams) 3 ml로 교체하였다.
tRNA의 존재하에 형질감염시킨 2개 웰 중 1개의 웰에서, 형질감염 8일 후에, 전형적인 세포 변성 효과가 있는 세포(둥근, 굴절성 세포)가 검출되었다. 이 세포의 상층액내 바이러스 분비는 qRT-PCR로 확인하였다. 형질감염 11일 후에, 단지 상기 웰에서만 현저한 세포 변성 효과가 검출되었으며, 나머지 3개의 형질감염을 수행한 웰의 상층액에서는 검출되지 않았다.
형질감염 후 회수한 바이러스 용액은 (16681 PDK50/Vero-2 대신) TV100이라 명명하였으며, 냉동보호제가 포함된 완충화된 수용액(pH = 7.5)로 1/2 희석한 후의 감염 역가는 5.8 log TCID50/ml이다.
1.3
형질감염 후 회수한 바이러스의 특정화
약독화에 중효한 특이 부위의 점 서열분석을 R. Kinney(CDC, Fort Collins)에서 수행하였다. 그 결과는 표 3에 나타낸다.
형질감염된 바이러스의 약독화 특이 위치의 서열분석
| 바이러스 | 5'NC-57 Nt57 |
NS1-53 Nt2579(aa) |
NS3-250 Nt 5270(aa) |
| DEN-2 16681 | C | G(Gly) | A(Glu) |
| DEN-2 PDK53 | T | A(Asp) | T/A(Val/Glu) |
| TV100 | T | A(Asp) | A(Glu) |
VDV2는 5'NC-57 및 NS1-53에 중요한 약독화 부위와, PDK53 백신에서 NS3-250-Glu 변이체의 야생형 16681 부위를 유지하고 있다. PDK53 백신에서 NS3-250- Glu/Val 혼합은 계대 배양물 PDK45 및 PDK53 간에 안정적인 것으로 관찰되어, Vero 세포에서 선택되는 것으로 제안되었다. 백신 접종자의 혈청으로부터 분리된 DEN-2 백신의 사전 분석으로, 이러한 선별 역시 인간에서 이루어질 수 있는 것으로 입증되었다.
바이러스 플라그의 직경은 Vero 세포에서 측정하였다. 간략하게, Vero 세포를 FBS를 4% 포함하는 배양 배지중에 밀도 1,000,000 cells/cm2로 접종하였다. 철야 배양한 후, 배지를 제거하고, 세포에 바이러스 2배 또는 5배 연속 희석물을 감염시켰다. 1.5시간동안 37℃, 5% CO2에서 배양한 후, 접종원을 제거하고, 세포를 1.26% 메틸셀룰로스 및 10% FBS가 포함된 최소 이글 배지(MEM)에서 37℃, 5% CO2에서 배양하였다. 11일간 배양한 후, 플레이트는 -20℃의 차가온 아세톤내에 20분간 방치하고, 2.5 ㎍/ml으로 희석시킨 플라비바이러스 특이 mAb를 이용하여 면역발색시켰다. 바이러스 플라그는 이미지 분석 소프트웨어(Saisam/Microvision)를 이용하여 측정하였다.
VDV2를 LAV2 16681/PDK50 종균과 비교하였고(표 4), 이는 1-3 mm 직경의 유사한 동질적인 소형 플라그를 나타내었다.
LAV2 16681/PDK50 및 VDV2의 플라그 크기
| 단계 바이러스 | LP/MP SP |
| 형질감염 전 LAV2 PDK50 형질감염 후 클로닝되지 않은 VDV2 |
0 319 0 183 |
LP/MP: 6웰에서 대형/중형 플라그의 수
SP: 6웰에서 소형 플라그의 수
1.4
플라그
-정제
추가적인 3회 증폭 계대 배양(P2 -> P4)를 형질감염 후 회수한 바이러스를 대상으로 수행하였다. 플라그-정제에 의한 생물학적 클로닝을 P3 및 P4 계대 배양한 바이러스(각각 LST 003 및 LST 007이라 명함)를 대상으로 수행하였다.
간략하게는, Vero 세포를 6웰 플레이트에 접종하고, 플레이트에 1 내지 20개의 플라그를 얻기 위해 바이러스 연속 희석물을 감염시켰다. 37℃, 5% CO2에서 1.5 시간 후에, 접종원을 제거하고 세포를 42℃에서 미리 열처리된 MEM-10% FCS로 이루어진 고형 배지를 42℃의 용해된 2% 아가와 즉시 혼합한 배지 3 ml하에서 배양하였다. 배지는 실온에서 플로우 후드 하에서 30분간 굳게 두었고, 플레이트는 뒤집은 상태로 10일간 32℃ - 5% CO2에서 배양하였다. 0.01% 중성 레드가 보충된 동일 배지의 두번째 층을 적층한 다음, 플레이트를 32℃에서 하룻밤 더 배양하였다. 잘 고립되어 있는 작은 플라그 6개를 0.1 ml 팁이 장착된 마이크로파이펫을 이용하여 멸균 조건하에서 취하여, 0.2 ml의 MEM-4% FCS가 있는 멸균 튜브로 옮겼다: P3 계대에서 3개(클론 31, 32 및 33)과 P4 계대에서 3개(클론 71, 72 및 73). 이를 동일 배지로 연속 희석하여 혼합함으로써 현탁액을 균질하게 한 다음, 이를 바로 Vero 세포의 6-웰 플레이트 감염에 사용하였다. 프로토콜을 반복하여, 2개의 SP를 클론 32, 33, 71 및 72에서, 하나의 SP를 클론 31에서 두번째로 취하였다. 취한 각각의 플라그는 배지 1 ml에 희석하고, T25 cm2 플라스크에서 Vero 세포 증폭을 수행하였다. 감염 6일째에 배양 배지를 취하여, 동량의 냉동보호제가 함유된 완충화된 수용액(pH 7.5)으로 희석하고, -70℃로 냉동시켰다. 상기 단계 모두 32℃에서 수행하였다.
플라그-정제된 바이러스는 각각 311, 321, 322, 331, 332, 341, 342, 351, 352, 711, 712, 721 및 722라고 명명하였다.
감염 역가는 1차 증폭이 끝난 시점에 Vero 세포에서 결정하였다(하기 참조):
클론 311: 3.95 LogCCID50/ml
클론 321: 5.20 LogCCID50/ml 클론 322: 5.45 LogCCID50/ml
클론 331: 5.55 LogCCID50/ml 클론 332: 4.95 LogCCID50/ml
클론 341: 2.80 LogCCID50/ml 클론 342: 4.85 LogCCID50/ml
클론 351: 5.35 LogCCID50/ml 클론 352: 5.50 LogCCID50/ml
클론 711: 5.45 LogCCID50/ml 클론 712: 5.65 LogCCID50/ml
클론 721: 5.30 LogCCID50/ml 클론 722: 5.60 LogCCID50/ml
Vero 세포에서의 이차 증폭을 3개의 클론: 클론 331, 352 및 722로 수행하였다. 배양 상층액을 형질감염 8일 후에 모아 냉동보호제가 포함된 완충화된 수용액(pH7.5)로 1/2 희석하여, TV331, TV352 및 TV722라고 명하였다.
1.5
클로닝된
바이러스의 특정화
1차 증폭 후, 증폭된 바이러스 모두 동일한 플라그 크기 표현형을 보였으며, (현저하게 낮은 크론 311 및 341을 제외하고는) 5 logCCID50/ml 또는 그 이상의 역가를 나타내었다. 1차 증폭한 6개의 클론(클론 321, 331, 351, 352, 711, 721)과 2차 증폭한 클론 3개를 대상으로, 약독화-특이 위치에 대한 서열분석을 수행하였으나, 돌연변이는 발견되지 않았다.
클론들간에도 어떠한 현저한 차이가 없어, VDV2 백신 후보 균주를 제조하기 위해, TV722를 선택하여 Vero 세포에서 증폭시켰다.
DEN-2 바이러스의 약독화 특이 위치의 서열분석
| 5'-UTR | prM | E | NS1 | NS2a | NS3 | NS4A | NS5 | |||
| 단계/세포 | 바이러스 | 57 | 524 | 2055 | 2579 | 4018 | 5270 | 5547 | 6599 | 8571 |
| 야생형/PGMK | 16681 | C | A | C | G | C | A | T | G | C |
| 백신/PDK | PDK53 | T | T | T | A | T | A/T | C | C | T |
| 이차 플라그 정제/Vero | TV321 | T | T | T | A | T | A | C | C | T |
| TV331 | T | T | T | A | T | A | C | C | T | |
| TV342 | T | T | T | A | T | A | C | C | T | |
| TV352 | T | T | T | A | T | A | C | C | T | |
| TV711 | T | T | T | A | T | A | C | C | T | |
| TV722 | T | T | T | A | T | A | C | C | T | |
| 2차 증폭/Vero | TV722PM | T | T | T | A | T | A | C | C | T |
뉴클레오티드 위치는 각 유전자 하기에 나타내며, DEN-2 16681 균주의 공개된 서열을 참조한다.
결론적으로, Vero 세포에서 순응된 DEN-2 16681/PDK50의 생물학적 클론을 수득하는데에는 총 11번의 계대 배양이 필요하였다.
또한, VDV2의 11회 계대 배양주의 전체 서열을 결정하고 표현형을 확인하여, 특정화를 수행하였다.
실시예
2: 서열분석
하기 전략에 따라 바이러스의 전체 서열을 제조하였다. 바이러스 게놈 RNA를 정제하였다. 전체 게놈은 16번의 중첩성 RT-PCR 반응에 의해 증폭하였다. 각 PCR은 서열분석 테그가 각 DNA 가닥에 부가되도록 설계하였다. 이는 보편적인 서열분석 프라이머 한쌍에 의해 수행되는 간단한 서열 반응을 가능하게 한다. 각 PCR 산물을 양쪽 DNA 가닥에 대해 먼저 서열분석하였다. 모든 결과를 조합하여, 전체 VDV2 게놈을 재구축하였다.
2.1
재료
2.1.1
바이러스
본원에서 참조되는 바이러스는 DEN-2 16681; LAV-2/PDK53; VDV2이며, 이들의 서열은 첨부된 서열 목록에 기재되어 있다.
이들 바이러스의 전체 게놈 서열은 10723개의 뉴클레오티드로 이루어져 있다.
2.1.2
프라이머
모든 프라이머는 프라이머 디자인 모듈인 Seqweb bioinformatics package (Accelrys)로 설계하였다(표 6)
RT-PCR 및 프라이머 서열 리스트
2.2
방법
2.2.1
바이러스
RNA
정제
이전의 실험에서, 다음 단계에서 RT-PCR 양성 반응을 얻는데에는 최소 1000 DICC50이 필요하였다. 이는, 바이러스 최소 역가 104 DICC50/mL이 필요하다는 것을 의미한다. 바이러스 게놈 RNA를 제조사의 권고안에 따라 QIAamp 바이러스 RNA 미니 키트(Qiagen)로 정제하였다. 간략하게는, 바이러스 조 샘플 중 140 ㎕을 라이시스 용액 존재하에 배양하여, 키트 컬럼에 주입하였다. 세정한 다음, RNAse 저해제(RNAse Out, Sigma) 1 ㎕(40 units)가 함유된, 뉴클레아제 결핍성 멸균수 60 ㎕로, 정제된 바이러스 RNA를 용리시켰다.
2.2.2
역전사
바이러스 RNA를 ABGene사의 역전사효소(reverse iT)를 사용하여 cDNA로 역 전사하였다. 즉, 10 ㎕의 정제된 RNA를 최종 반응 부피 20 ㎕로 표준 조작 조건을 적용하였다. 반응은 (-) 가닥 프라이머의 혼성화로 개시하였다. PCR 당 한번의 RT 반응을 수행하였다. cDNA 합성은, 47℃에서 45분간 인큐베이션하여 수득하였다.
2.2.3
PCR
모든 PCR은 표 6의 16쌍의 프라이머 (+) 및 (-)를 이용하여 연장된 고정확성 PCR 시스템(Expand High Fidelity PCR system, Roche diagnostics)으로 수행하였다:
RT 2㎕
10x 완충액 2.5㎕
dNTP mix (10mM) 2㎕
프라이머 각 0.8㎕
물 16.4㎕
효소 0.5㎕
PCR
프로그램
변성: 94℃, 2 분 -> (변성: 94℃, 15 초 -> 혼성화: 55℃, 30 초 -> 연장: 68℃, 1 분): 40회 -> 연장: 68℃, 5 분
모든 PCR 산물은 아가로즈 젤상에서의 전기영동으로 관리되었다.
2.2.4
서열분석
서열분석 반응 대부분은 Genome Express 사에서 실시하였다. 게놈 양극단, 모호한 부분, 일부 인터-PCR 정션 및 기술적인 이유로 Genome Express에서 분석되지 않는 영역은 사내에서 수행하였다.
- Genome Express에서의 서열분석: PCR 산물은 +4℃에서 취급하였고, 서열분석 결과는 서열 정보 파일로 수득하였다. 텍스트 파일, 정성 파일 및 크로마토그램은 각 개별 서열에 대해 이용가능하다. 서열 정렬을 수행한 후, 크로마토그램상에서 일치되지 않는 부분을 모두 체크하고, 서열 알고리즘 에러로 파악되는 것은 보정하였다.
- 사내 서열분석: 서열 반응은 Sequitherm Excell II LC kit (Epicentre)를 이용하여 써모사이클러 PTC-200(MJ Research)으로 수행하였다. 각 PCR 산물은 단일 반응으로 독립적으로 양쪽 가닥을 서열 분석하였다. 반응물은 서열 전기영동 젤에 로딩하였다. 전개 및 서열 분석은 자동 서열분석기인 Gene ReadIR 4200 (Li-Cor)에서 수행하였다.
- 서열분석 반응물
DNA 최대 200/250 ng
반응 완충액 7.2㎕
프라이머(1-2 pM) 각 1.5㎕
효소 1㎕
물 최종 부피 20㎕으로 잔량 첨가
PCR
프로그램
변성: 92℃, 2분 -> (변성: 92℃, 15 초 -> 혼성화: 50℃, 30 초 -> 연장: 70℃, 1 분): 30회 -> 연장: 70℃, 10 초
변성/로딩 완충액 3 ㎕을 첨가한다.
샘플의 변성은 95℃에서 3분간 수행하고 샘플 로딩 전까지 얼음에서 냉각시킨다.
-서열분석을 위한 전기영동
전기영동 파라미터:
전압 1500 V
전류 35 mA
전력 40 W
수행 시간 9시간
젤 파라미터:
젤의 길이 41 cm
젤의 두께 0.2 mm
온도 45℃
스캔 속도 3
2.3
결과
모든 PCR 단편은 테일이 부가된 일반 PCR, 즉 모든 프라이머의 5' 말단에 부가된 특이적인 모티프를 이용하여 양 말단으로부터 서열분석하였다.
5' 프라이머: M13SEQ-GTTTTCCCAGTCACGAC (서열번호 36)
3' 프라이머: M13REV-AACAGCTATGACCATG (서열번호 37)
M13-SEQ 및 -REV 서열은 보편적인 M13 프라이머 모티프(New England Biolabs references)에 상응한다.
마지막 단편 조립(contig assembly)을 위해, ContigExpress module (Informax)의 Vector NTi에서 신속한 분석을 수행하였다. LAV2 기준 서열을 각 서열분석 결과 모두와 비교하였다. 그 결과, 일부 영역에 단편 조립이 여전히 없을 경우에도 모든 결과는 전체 게놈에서 우측으로 정렬되었으며, 이는 전체 게놈 정렬을 신속하게 보여준다.
2.3.1
완전한
VDV2
서열 조립
최종 서열 정렬을 Vector NTi, AlignX module (Informax)에서 수행하였다. 고전적인 다중 서열 정력 알고리즘 ClustalW(Thompson et al., 1994)을 소프트웨어로 구동하여, 전체를 정렬하였다. 모든 서열 결과를 LAV2 기준 서열과 함께 정렬하였고, 그 결과 게놈을 보다 잘 재구축할 수 있었다. 기준 서열에 대해 나타나는 모든 서열 차이는 다른 독립적인 서열 반응으로 검증하여야 한다. VDV2의 전체 서열은 서열번호 1로 나타낸다.
일부 모호한 부분이 단일 서열, 특히 양 말단 근처 서열에서 나타났다. 이는 PCR 단편의 양 말단에서의 일부 반응 불량이 본래 있는 곳이다. 이러한 정성적으로 불량한 서열은, 다른 2가지 서열 반응을 다른 PCR 산물로부터 입수가능할때까지, 정렬에서 제외시켰다. 적어도 2개 이상의 독립적인 다른 PCR 서열에서 일치되는 것으로 확인되지 않는 경우에는, 기준 서열과의 차이나는 부분은 최종 정렬에 참작하지 않았다. 역으로, 2개의 독립 서열에서 확인된 모든 차이는 최종 서열에 나타내었다.
표 7은 각 개별 서열 반응의 특징을 총괄한 것으로, 개시부, 종결부 및 길이를 표시하고 있다. 인접한 PCR 산물간의 중첩과 기준 서열과의 차이를 마지막 열에 나타낸다.
뎅기 VDV2의 개별 서열 특징
| 명칭 | 개시 | 종결 | 크기 | 중첩 | 코멘트 |
 |
|||||
cDNA 합성 및 PCR DNA 반응에 게놈 말단에 상보적인 올리고뉴클레오티드가 필요하기 때문에, 게놈의 양 말단은 PCR 증폭으로 서열분석할 수 없었다. 증폭 단계 동안에, 이들 올리고뉴클레오티드를 PCR 단편으로 병합하였다. 서열 결과는 합성 올리고뉴클레오티드의 결과이며, 바이러스 그 자체의 서열 결과는 아니다. 바이러스 게놈의 양 말단으로부터의 PCR은 적절하게 수행되었는데, 이는, 바이러스 서열이 올리고뉴클레오티드 서열과 크게 다르지 않음을 시사한다(크게 다를 경우, PCR 증폭은 실패하거나 적어도 불량하게 수행된다). 본 발명자들은 이들을 다른 PCR 증폭산물과 구별할 수 없었다. 그래서, 재구축한 게놈에서, 게놈의 양쪽 말단을 올리고뉴클레오티드 서열과 동일한 것으로 간주하였다(그리고, 기준 서열과 동일한 것으로 간주함). 5' 말단의 서열은 뉴클레오티드 1 - 32이다. 3' 말단의 서열은 뉴클레오티드 10695-10723이다.
2.3.2
서열 비교
모균주 LAV2 게놈 서열에 대해 10개의 뉴클레오티드가 상이한 것으로 확인되었다. VDV2 백신 균주는 바이러스 위생 처리 및 개에서 원숭이 세포로의 계대 배양을 통해 LAV2로부터 파생된 것이다.
LAV2과 VDV2 간의 차이 발생에는 여러가지 이유가 있을 수 있다. 첫째, 클로닝 단계에서, LAV2에서 이미 검출된 다수의 서열과 100% 동일하지 않는 바이러스 하위집단이 선정될 수 있다. 둘째로, LAV2는 PDK 세포에서 생산되지만, VDV2는 Vero 세포에서 만들어진다. 이러한 개에서 원숭이 세포로의 계대 배양은 새로운 세포주에 순응하도록 바이러스 변화를 선호적으로 유도한다. 세번째로, 모든 RNA 바이러스의 경우, 정확성(fidelity)이 보다 낮은 바이러스의 RNA 중합효소는 DNA 중합효소에 비해 게놈에 돌연변이를 일으킬 확률이 더 높다.
LAV2 바이러스의 약독화와 연결되어 있는 9개의 뉴클레오티드 위치 모두, 서열이 VDV2 계대 배양 11 균주에 보존되어 있었다.
또한, VDV2 계대 배양 9회 균주 및 11회 세대 배양 균주간의 서열 비교로, 11회 계대 배양주 및 9회 계대 배양주 간에, 표현형, 바이러스 혈증 및 면역성 차이와 연결된 2개의 돌연변이가 확인되었다.
LAV2/PDK53 균주와 VDV2 11회 계대 배양주 및 9회 계대 배양주 간의 서열 비교
| 뉴클레오티드 | 아미노산 | |||||
| 위치 | LAV2 | VDV 9회 계대 11회 계대 |
부위 | 위치 | LAV2 | VDV 9회 계대 11회 계대 |
 |
||||||
볼드체: VDV 9회 계대와 11회 계대 균주간에 서열이 상이한 부분
이용가능한 모든 Genbank 혈청형 2의 뎅기 게놈 서열들간에 서열 정렬을 수행한 경우, 단지 2곳만 다른 뎅기 2 균주(1638 및 2520)와 공유되며, 이 2곳 모두 아미노산 수준에서는 침묵으로 나타난다(표 8). 아미노산 변동과 관련하여, 비구조 펩티드에서의 4개의 변동은 생화학적 관점으로는 비교적 보존적으로 것으로 보이나, M 및 엔벨로프에서의 2개의 변동은 전하 및 소수성 모두에 변화를 가져올 것으로 보인다.
실시예
3: 특정화
본 연구의 목적은 계대 배양을 통해 약독화 마커에 변화가 있는지를 분석하기 위한 것이었다.
도 2에 나타낸 플로우 차트는 Filled Product(일가), "즉시 사용가능한" 투약제를 만드는 개발된 제조 공정을 요약한 것이다.
간략하게, VDV2 8회 계대 배양 균주를 Vero 세포에서 2회 연속 계대 배양한 후, 해당 사용(working) 종균을 수득하였다. 최종 바이러스 배양은, Vero 세포 현탁물의 감염에 의해 수행되었다. 생산된 바이러스를 회수하였다. 데옥시리보뉴클레산(DNA)를 효소 처리로 분해시켰다. 불순물은 초여과로 제거하였다. 감염 역가는 농축 단계에 의해 증가시켰다. 냉동보호제를 포함하는 완충화된 수용액(pH= 7.5)을 첨가하고, 이 0.22 ㎛ 여과한 혼합물을 동일 용액내 표적 투여량으로 희석하였다. 이후, 활성 물질은 유리 바이얼에 넣고, 냉동 건조하여, 사용전까지 보관하였다.
3.1
표현형
마커
표 9는 DEN-2 16681 wt 균주, DEN-2 16681/PDK53 백신 균주, VDV2 9회 계대 배양 균주 및 VDV2 11회 계대 배양 균주(최종 순응된 계대)를 대상으로 실시한 3번의 표현형 분석의 결과를 나타낸다: 온도-민감성(Ts), 원숭이(Vero) 및 모기(C6/36) 세포에서의 증식 곡선 및 마우스 신생아에서의 신경 독성(CDC에서 수득한 결과). 마우스 신경 독성 감소(사망율 감소 및 평균 생존 시간(AST) 연장), 39 ℃에서의 제한적인 증식 및 C6/36에서의 제한적인 복제는, 뎅기 바이러스에 대한 약독화 기준으로서 과학계에서 현재 수용하고 있다. Vero-순응된 계대 균주는 명확한 Ts 프로파일을 나타내며, DEN2/PDK53 보다 더욱 제한적이다. 최종적으로 순응된 계대 균주는 본 분석에서 약 3 log로 제한된다. 온도 민감성 역시 바이러스 증식 곡선으로 검증되었다. Vero 세포에서 테스트한 모든 바이러스에서, 유사한 복제율이 관찰되었다. 모기 세포에서는, Vero-순응된 바이러스의 증식은 wt DEN2에 비해 명확하게 제한되었으며, DEN2-PDK53과 비교하여 약간 제한적(약 0.5 log)이었다. 놀랍게도, Vero-순응된 바이러스의 마우스 신경독성은 wt DNE2의 신경독성에 가까웠고, DEN2/PDK53 백신의 신경독성 보다 현저하게 높았다. 이러한 데이타는, 바이러스 균주의 약독화 측면에서 이의 예측치가 낮음을 의미한다(임상 데이타와 비교).
VDV2 9회 및 11회 계대 균주, DEN2/PDK53 및 wtDEN2의 플라그 크기 분포를 도 5에서 비교하였다. wt DEN2는 플라그의 95%가 사이즈가 동질하며 플라그 다수 집단(81%)의 크기가 < 0.6mm이고, 소수(12%)가 1-2 mm인, 혼성 프로파일을 보인다. 이러한 프로파일은 DEN2/PDK53의 프로파일에 가깝지만, 구별되었다. 순응의 중간 계대 균주인 VDV2 P9는 다수 집단(70%)의 플라그 크기가 1-2 mm이고 소수(25%)가 < 0.6 mm인, 혼성 프로파일을 보인다. 이들 데이타는, VDV2 균주가 P9에서 완전히 순응되지 않았음을 나타내며, Vero 세포에서 안정적으로 복제되는 동질적인 집단을 수득하기 위해서는 2번의 부가적인 계대 배양이 필요함을 의미한다.
DEN-2 바이러스의 표현형 요약
| 온도 민감성 (39℃에서의 역가 감소율%)Fold - reduction |
증식 곡선(peak log10 pfu/ml) Vero-LS10 |
신생 Swiss Webster 마우스에 대한 신경독성 | |||||
| 바이러스 | 스코어 | 3일 | 4일 | 5일 | 6일 | 역가 일 | 사망율n AST(S.D.) |
| D2-16681 | + | n.d. | 92.7 13.7 | n.d. | 92.2 12.8 | 7.5 8 | 100.0%16 12.2(1.5) |
| D2-PDK53 | + | n.d. | 96.6 29.4 | n.d. | 99.7 333.3 | 7.3 8-10 | 43.75%16 16.0(2.4) |
| VDV2 P9 | + | n.d. | 99.94 1666.7 | n.d. | 99.97 3333.3 | 7.5 8-10 | 100.0%16 10.9(0.7) |
| VDV2 P11 | + | n.d. | 99.92 1250.0 | n.d. | 99.88 833 | 7.5 10 | 100.0%16 10.9(0.6) |
*n: 동물의 수
실시예
4:
원숭이에서의
면역성, 바이러스
혈증
및 독성
원숭이에게서 수득할 수 있는 대부분의 실제 수많은 데이타는 면역성 및 바이러스 혈증에 대한 것이다. 특히, 바이러스 혈증은 인간에서의 유독성 및 질환 중증도과 관련있는 인자들중 하나로서 식별되고 있으며, 고려할 중요 파라미터이다. 명확하게는, 면역성은 백신 테스트시 중요한 파라미터이다.
본 발명자들은 바이러스 혈증 및 면역성에 대한 최저/최대값을 확립하였다.
플라그 분석에 의해 Vero 또는 LLC-MK2 세포에서 측정된 바에 따른, 원숭이에서 뎅기 백신 후보물질에 의해 유도된 반응에 대한 최소 요건(이들 세포는 이러한 분석에서 등가로 간주됨)
| 바이러스 혈증의 평균 지속 기간 (일) (모든 혈청형을 고려함) |
바이러스 혈증의 평균 최고 역가 (log 10 pfu) (모든 혈청형을 고려함) |
평균 중화 역가 30 일 (각 혈청형에 대해) PRNT 50 |
| ≤ 3 일 | ≤ 1.5-2 | ≥ 80 |
pfu: 플라그 형성 단위
PRNT 50: 플라그 감소 중화 역가 50(플라그 갯수의 50%를 감소시키는 역가)
4.1
동물에서의 전-임상 약리학, 약동학 및 산물 대사
4.1.1 재료 및 방법
4.1.1.1 원숭이 실험
원숭이 실험을 유럽의 동물 실험 가이드라인에 따라 수행하였다.
면역화는 Mauritius(CRP Le Vallon)에서 기원된 시노몰러스 원숭이(Macaca fascicularis)를 대상으로 수행하였다. 원숭이는 면역화하기 전에 Sanofi Pasteur의 동물 실험실에서 6주간 격리시켰다.
원숭이의 팔에 백신 0.5 ml을 피하(SC) 경로로 백신 접종하였다(해당 섹션 참조). 케타민(Imalgene, Merial)으로 간단하게 마취시킨 후, 서혜부 및 복재 정맥의 천공에 의해 채혈하였다. 0일 및 28일째에, 혈액 5 ml을 항체 반응 평가를 위해 샘플링하였고, 2일 내지 10일 사이에 혈액 1 ml은 바이러스 혈증 평가를 위해 샘플링하였다. 혈액을 얼음 위에서 모은 다음 혈청이 분리될때까지 얼음 위에 두었다. 혈청 분리를 위해, 혈액을 20분간 4℃에서 원심분리하고, 혈청을 채집하여 Rich Kinney's laboratory에서 테스트할 때까지 -80℃에 보관하였다. 미국으로 이송시 드라이아이스에서 유지시켰다.
4.1.1.2 바이러스 혈증 및 중화 항체 반응(플라그 감소 중화 테스트, PRNT)
모든 분석은 미국의 포트 콜린스 CDC의 R. Kinney 실험실에서 수행하였다. 혈청 샘플을 -80℃로 유지하여 이송시키고, 테스트할 때까지 상기 온도로 저장하였다. 일차 해동시, 샘플은 바이러스 혈증에 대해 테스트하였고, 혈청을 1:5로 희석시켰다. 1:5 혈청 희석물은 중화 항체 테스트하기 전에 56℃에서 30분간 불활화하였다.
4.1.1.2.1 바이러스 혈증
0.125 ml의 혈청을 96웰 플레이트의 첫번째 웰내 희석물 0.125 ml(RPMI medium)에 넣고, 각 희석으로 0.025 ml을 0.225 ml로 이동시켜 10배 연속 희석을 수행하였다. 0.2 ml의 100.3-105.3 연속 희석물을 6웰 플레이트의 Vero 세포에 넣었다(바이러스는 37℃에서 1.5시간동안 흡착시키고, 아가로스가 무첨가된 중성 레드 4 ml을 적층한 다음 중성 레드가 함유된 아가로스 2 ml을 6-7일 후에 적층하여, 플라그의 수를 측정함). 바이러스의 검출 한계는 10 PFU/ml이었다. 대조군으로, 스톡 DEN-16681 PDK-13 (LAV2) 백신을 접종하였다.
4.1.1.2.2 PRNT(플라그 감소 중화 테스트)
중화 항체를 Huang et al.(2000)에 개시된 바에 따라 정량하였다. 간략하게는, 0.2 ml의 열에 의해 불활화한 1:5 혈청 희석물을 96웰 플레이트의 첫번째 웰에 넣고, 각 희석으로 0.1 ml을 희석물(RPMI 배지) 0.1 ml에 이동시켜 2배 연속 희석을 수행하였다. 그 결과 1:10 - 1:320의 혈청 연속 희석물이 제조되었다. 0.1 ml의 DEN 바이러스(60-160 PFU; 모균주 DEN2 16681 바이러스)를 혈청-바이러스 혼합물의 최종 부피 0.2 ml로 각 혈청 희석물이 있는 웰에 첨가하였다. 96웰 플레이트는 4℃에서 철야 배양하였다. 0.1 ml의 혈청-바이러스 혼합물(첨가한 바이러스가 30-80 PFU로 포함됨)을 6웰의 Vero 플레이트에 접종하고(바이러스 혈증 섹션에 기재된 내용 참조), 중성 레드로 염색하여 플라그 수를 측정하였다. 2배, 1배 및 0.5배 테스트 농도로 첨가한 바이러스의 여러가지 백 역가 확인(multiple back titration)으로, 50% (PRNT50) 및 70% (PRNT70) 종말점 항체 역가를 결정하는 기준인, 주입 PFU의 직접적인 실험치를 결정하였다. 네거티브 혈청 결과는 중화 항체 역가가 < 1: 10이어야 한다. 중화 역가 320을 보이는 혈청은 종말점 역가 결정을 위해 1:80 - 1:2560 희석물에서 재분석하였다.
4.1.2
원숭이에서의
9회
계대한
일가
VDV2
후보물질의 평가
VDV2 후보물질의 정제/선정은 실시예 1에 기재된 바에 따라 수행하였다. (표현형 마커 및 서열을 토대로) 선정된 클론을 9회 계대 배양한 후, Sanofi pasteur에서 CRP Le Vallon, Mauritius 기원의 시노몰구스 마카크 수컷(Macaca fascicularis, 평균 체중 3.1 kg)을 대상으로 테스트하였다.
D0에 면역화한 다음, D2 내지 D10 동안 바이러스 혈청을 조사하였고, D0 및 D28에 면역성을 측정하였다. 바이러스 및 백신은 액체 형태로 사용하는 경우 모두 -70℃에서 유지시켰다.
LAV2 : 역가 : 103,93 DICC50 / ml; 동결건조하고, 0.5 ml의 PBS(Ca2 + 및 Mg2+; CaCl2.2H2O 0.133 g/l; MgCl2.6H2O, 0.1g/l 함유)에 재현탁한 다음 모두 투여함.
계대 배양 VDV2 DEN2-TV722(2회 플라그 정제 + 1회 증폭) : 역가 : 105,6 DICC50 /ml; 액체, PBS(Ca2 + 및 Mg2 +; CaCl2.2H2O 0,133 g/l; MgCl2.6H20, 0,1g/l 함유)에 105,3 pfu/ml로 희석하고, 0.5 ml 투여함.
23G1 바늘로 VDV2를 105 DICC50 투여량으로 팔에 피하 주사하였다.
결과는 표 11에 나타낸다. 28일째의 역가 확인은 PRNT70 및/또는 PRNT50을 3세트로 수행하였다.
VDV2와 LAV2 비교시 바이러스 혈증에 대해 명확한 차이가 나타났으며, 3/4 원숭이에서 VDV2의 단기간의 바이러스 혈증은 LAV2 보다 높았으며, 양쪽 타입에 대한 면역성이 현저하였다(VDV2에 대해 다소 낮음). 이러한 VDV2의 바이러스 혈증은 Vero 세포에서 수회 계대 배양한 후 프리-마스터 수준에서는 너무 높은 것으로 여겨질 수 있다. 그러나, 야생형 DEN2(및 그외 타입들 역시) 보다 장기간(6-7일간)동안 바이러스 혈증을 유도하며, 밀도도 높다(최대 5 log pfu)(Monath et al., 2000; Bray et al., 1996).
VDV2의 9회 계대 균주의 면역성
4.1.3
11회 계대 배양한 일가
VDV2
후보물질의 평가
백신의 면역성을 9회 계대 배양 균주를 대상으로 수행하였기에, 2회 더 계대 배양한 후(10회 계대) 일가 계대 배양주를 테스트하기 위한 실험을 또한 설계하였다.
C.R.P. Le Vallon, Ile Maurice 기원의 마카카 파시큘라리스(Macaca fascicularis) 원숭이 수컷을 상기와 같이 사용하였다(24마리의 원숭이, 평균 체중 3.4 kg).
VDV2 의 11회 계대 배양 균주; 배치: 역가 : 8.07 log10 DICC50/ml
위약: Ca2 + 및 Mg2 +이 포함된 PBS
VDV3: Vero 유래 백신 뎅기 혈청형 3 균주, Vero 세포에서 LAV3의 위생 처리에 의해 수득
VDV4: Vero 유래 백신 뎅기 혈청형 4 균주, Vero 세포에서 LAV3의 위생 처리에 의해 수득
백신을 (Ca2 + 및 Mg2 +; CaCl2.2H2O 0.133 g/l; MgCl2.6H2O, 0.1g/l 포함된) PBS에 105.3 DICC50/ml로 희석하고, 23G1 바늘로 팔에 투여량 105 DICC50에 해당되는 0.5 ml을 투여하였다.
바이러스 혈증 및 면역성을 R Kinney에 의해 CDC에서 측정하였다. 그 결과는 표 12에 나타낸다.
VDV2의 11회 계대 배양 균주의 일가 백신은 현저한 면역 반응을 유도하지만 바이러스 혈증은 낮거나 없었다. 이러한 없거나 낮은 VDV2 유도성 바이러스 혈증은 VDV2의 9회 계대 배양 균주가 매우 초기에 바이러스 혈증을 유도하였던 이전 실험 측면에서 고려된다. 9회 계대와 11회 계대 사이의 이러한 진화는 높은 바이러스 혈증을 억제시켰지만, 면역성은 유지시켰다. 따라서, VDV2는 수용가능한 후보 물질이다.
동일한 실험에서, 원숭이 4마리에 동일한 VDV2의 11회 계대 백신이 포함된 4가 제형을 백신 주사하였지만, VDV1 및 VDV2에 대한 바이러스 혈증은 검출되지 않았고, VDV3과 VDV4는 바이러스 혈증을 유도하였다.
다른 2번의 실험에서는, VDV2를 단독으로 또는 다른 혈청형과 조합하여 사용하였다.
첫번째 실험에서(4가 실험; 각 혈청형의 5 log), VDV2 및 VDV1에 대한 바이러스 혈증은 검출되지 않았지만, VDV3 및 VDV4에 대해서는 높은 수준의 바이러스 혈증이 검출되었다.
두번째 실험에서, VDV2의 11회 계대 배양주를 단독으로 또는 VDV1을 포함하는 4가 조합물로 투여하였다. 단독 투여시, VDV2의 11회 계대 배양주는 원숭이 4마리중 한마리에서만 바이러스 혈증을 낮게(peak 40) 유도하였지만, 나머지 3마리는 바이러스 혈증이 나타나지 않았다. 4가 제형 투여시, 심지어 VDV2를 4 log로 투여하고 VDV3 및 VDV4를 2 log로 투여하더라도, VDV2는 바이러스 혈증을 유도하지 않거나, 또는 VDV3 및 VDV4에 비해 매우 낮게 유도하였다. 이 결과는, 바이러스 혈증 측면에서는 VDV2의 안정성이 높다는 것을 나타낸다. 따라서, 일가 VDV2는 원숭이에서 최초로 규정된 성공 기준을 만족시켰다.
VDV2의 11회 계대 배양 균주의 면역성 및 바이러스 혈증
4.2
VDV2
의 독성
4.2.1
원숭이에서의
신경독성
테스트
이 실험의 목적은 Sanofi Pasteur에서 생산된 약독화된 2 뎅기 바이러스 종균의 원숭이(Ph. Eur 2.6.18)에 대한 향신경성 특성이 없음을 입증하기 위한 것이다.
Mauritius 유래 시노몰구스 원숭이 10마리에 VDV2의 9회 계대 배양 균주를 뇌내 경로로 접종하였다(107.10CCID50/mL, 각 뇌 반구의 시상에). 테스트 종료시, 원숭이를 죽이고 포르말린 용액을 주입하였다. 각 원숭이의 뇌에서 조직 샘플을 취하였다(연수, 뇌교, 소뇌, 중뇌, 좌우 파트를 포함한 시상, 대뇌 피질의 좌 및 우). 조직 단편은 8 ㎛의 두께로 잘라 에오신과 갈로시아닌으로 염색하였다.
혈청형 2 프라이머리 바이러스 종균을 주입한 원숭이의 뇌에서는 어떠한 병원성을 보이는 조직병리학적 신호는 관찰되지 않았다.
4.2.2 VDV2 의 1회 피하 투여 후 28일간 관찰하는, 시노몰거스 원숭이에서의 GLP 독성 실험
이 GLP 실험의 목적은 VDV2의 9회 계대 배양 균주와 다른 뎅기 백신 후보물질 간에 상호작용을 평가하기 위한 것이다. 실험의 제1 단계는 다른 백신 후보물질을 투여하기 전에 VDV2의 안전성과 면역성을 평가하기 위한 것이다.
시노몰거스 원숭이(수컷 4마리, 암컷 4마리)에 0일에 VDV2를 인간 1회 투여량(1회 투약 당 약 104 CCID50)으로 피하 투여하였다. 수컷 2마리와 암컷 2마리로 이루어진 대조군 그룹에는 비히클(NaCl 4 ‰)을 제공하였다.
실험 전반에 거쳐 사망율, 임상 증상, 체중 및 음식 소모량을 모니터링하였다. 투여한 그날부터 매일 테스트하기 전에 그리고 투여 후 2일동안 매일 한번씩 체온을 측정하였다. 혈액 샘플을, 테스트 전, 8일 및 27일째에 임상 실험 측정을 위해 취하였다.
임상적인 증상, 체중, 음식 소모량, 피부 반응, 체온, 혈액학, 임상 화학 또는 장기의 무게에는 별 영향이 없었다. 실험하는 동안에 사망한 동물은 없었다.
결론적으로, 시노몰거스 원숭이(Macaca fascicularis)에 VDV2를 테스트 용량으로 피하 투여하였을때, 생전에 임상 관찰 결과 및 임상적인 병리로 평가한 바에 따르면, 원숭이의 건강에 나쁜 영향을 미치지 않았다.
실시예 5: 18 내지 40세의 플라비바이러스에 노출된적 없는 건강한 성인에서 의 일가 VDV2의 안전성
1상 실험의 목적은 플라비바이러스에 노출된적 없는 성인에서 Stamaril®(대조군)와 비교하여 104 CCID50의 바이러스 농도에서 1가 VDV2에 대한 안전성, 바이러스 혈증 및 면역성 프로파일을 확인하기 위한 것이다. 1회 주사한 다음 6개월 및 12개월간 추적하였다. 안전 예방책으로, 순차적 통합(sequential inclusion)을 수행하였다.
이에, 등록 및 백신 접종은 엇갈리게 수행하였다: 1차 코호트(그룹 당 n=4, 총 n=12)에 백신 접종하였다. 최대 28일째까지 수집한 안전성 데이타는, 나머지 개체(그룹 당 n=8, 총 n=16)에 대한 백신 접종 진행을 결정하기 전에 IDMC(Independent Data Monitoring Committee) 및 왕립 애덜레이드 병원의 IDSC(Investigational Drugs Subcommittee)에서 논의하였다. 실험 설계의 개략적인 사항은 도 6에 나타낸다.
백신을 투여한 후, 환자를 다양한 임상 실험 및 테스트로 정기적으로 진찰하였다. 그 결과는 하기 표 13에 나타낸다.
등록된 집단은 플라비바이로스에 노출된 적 없는 사람 [플라비바이러스 질환(예, 황열, 일본 뇌염, 뎅기열)에 대해 백신 접종을 받은 개체; 또는 플라비바이러스 감염 병력이 있는 개체(임상적으로, 혈청학적으로 또는 미생물학적으로 확인됨), 또는 이전에 뎅기 감염 풍토성이 높은 지역에서 (얼마 동안이라도) 거주 또는 여행한 적이 있는 개체, 또는 2주 이상동안 노스 퀸슬랜드에 거주 또는 여행한 적 있는 사람은 제외됨]을 포함하며, 18 내지 40세(즉, 18번째 생일인 날에서 41번째 생일이 되기 전날)의 성인으로 구성된다.
조사한 플로우 차트
| 진찰 횟수 | V01 | V02 | V03 | V04 | V05 | V06 | V07 | V08 | V09 | V10 | V11 | V12 |
| 실험 시간표 # | D0 | D2 | D4 | D6 | D8 | D10 | D12 | D14 | D16 | D28 | D180 | D365 |
| 시간 윈도우 | +1d | +1d | +4d | +15d | +30d | |||||||
| 임상 검사 | v | v | v | v | v | v | v | v | v | v | v | v |
| 바이탈 사인(BP, 맥박수) | v | |||||||||||
| 구강 온도 | v | v | v | v | v | v | v | v | v | v | ||
| 혈액 샘플링: -혈청 HBV/HCV/HIV -생물학적 안전성 -바이러스 혈증 -면역성 -혈청내 사이토카인 -T 세포(세브세트)에 대한 PBMC -국소 및 전신 현상의 직접 감시 |
v v v v v v v v |
v v v |
v v v v |
v v v |
v v v v |
v v v |
v v v v |
v v v |
v v v v v |
v v v v v v |
v v |
v v |
V: 진찰 - D: 일(day)
# 진찰 시간 간격은 진찰한 날과는 다른 백신 접종 실험한 날로부터 계산된다(예, 임시 배제 기준에 해당되는 경우). V06 및 V07은 적어도 1일 이상의 간격으로 행해져야 한다.
테스트한 제품은 다음과 같다:
조사된 백신은 별개로 제공된 희석제를 사용하여 즉석에서 재구성되는, 바이얼에 담기 동결건조된 제품;
활성 성분: 투여량 0.5 ml 당 각 1가 Vero 뎅기 바이러스 혈청형 2(VDV2) 4 + 0.5 log10 CCID50;
희석제: 백신 재구성을 위한 멸균 NaCl 4‰ 용액.
재구성된 백신, 즉 1가 VDV2의 NaCl 4‰ 용액 0.5 mL은 즉시 사용하거나 사용하기 전까지 +2℃ 내지 +8℃에서 유지시켜야 한다.
백신 투여량 0.5 mL은 삼각근에 피하 투여된다.
대조군 백신인 Stamaril®은 Aventis Pasteur 사에서 생산되는 황열 백신이다. Stamaril®은 동결건조된 형태로, 조류-로이코시스-결핍성, 안정화된 제품으로, 사용하기 바로 전에 희석제를 이용하여 재구성된다. (활성 성분: 약독화된 황열 생바이러스(17D 균주): >1,000 마우스 치사량 50% (LD50)/희석제: 멸균된 NaCl 4‰ 용액).
대조군 백신은 삼각근에 피하 투여된다.
개체는 백신 접종과 관련된 임상적으로 유의한 증후군을 나타내진 않았다. 개체 한명은 일과성 열(<38℃)이 발생하였다. 개체 한명은 국소 반응(경화)을 보였다. 백신 접종과 관려된 심각한 부작용은 관찰되지 않았다.
모든 개체는 뎅기 2에 대한 백신 접종 후 28일 경과시에 항체 반응성을 보였다(역가 1888 - 6393).
참조문헌
<110> Sanofi Pasteur
Centers for Disease Control and Prevention
<120> Dengue serotype 2 attenuated strain
<130> BET 06P0277
<150> US 60/691274
<151> 2005-06-17
<160> 42
<170> KopatentIn 2.0
<210> 1
<211> 10723
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10723)
<223> VDV2
<220>
<221> CDS
<222> (97)..(10272)
<400> 1
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctg atg aat aac caa cgg 111
Met Asn Asn Gln Arg
1 5
aaa aag gcg aaa aac acg cct ttc aat atg ctg aaa cgc gag aga aac 159
Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn
10 15 20
cgc gtg tcg act gtg caa cag ctg aca aag aga ttc tca ctt gga atg 207
Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg Phe Ser Leu Gly Met
25 30 35
ctg cag gga cga gga cca tta aaa ctg ttc atg gcc ctg gtg gcg ttc 255
Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe
40 45 50
ctt cgt ttc cta aca atc cca cca aca gca ggg ata ttg aag aga tgg 303
Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly Ile Leu Lys Arg Trp
55 60 65
gga aca att aaa aaa tca aaa gct att aat gtt ttg aga ggg ttc agg 351
Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val Leu Arg Gly Phe Arg
70 75 80 85
aaa gag att gga agg atg ctg aac atc ttg aat agg aga cgc aga tct 399
Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Arg Arg Arg Arg Ser
90 95 100
gca ggc atg atc att atg ctg att cca aca gtg atg gcg ttc cat tta 447
Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val Met Ala Phe His Leu
105 110 115
acc aca cgt aac gga gaa cca cac atg atc gtc agc aga caa gag aaa 495
Thr Thr Arg Asn Gly Glu Pro His Met Ile Val Ser Arg Gln Glu Lys
120 125 130
ggg aaa agt ctt ctg ttt aaa aca gag gtt ggc gtg aac atg tgt acc 543
Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly Val Asn Met Cys Thr
135 140 145
ctc atg gcc atg gac ctt ggt gaa ttg tgt gaa gac aca atc acg tac 591
Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr Ile Thr Tyr
150 155 160 165
aag tgt ccc ctt ctc agg cag aat gag cca gaa gac ata gac tgt tgg 639
Lys Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu Asp Ile Asp Cys Trp
170 175 180
tgc aac tct acg tcc acg tgg gta act tat ggg acg tgt acc acc atg 687
Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Met
185 190 195
gga gaa cat aga aga gaa aaa aga tca gtg gca ctc gtt cca cat gtg 735
Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val Pro His Val
200 205 210
cga atg gga ctg gag aca cga act gaa aca tgg atg tca tca gaa ggg 783
Arg Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly
215 220 225
gcc tgg aaa cat gtc cag aga att gaa act tgg atc ttg aga cat cca 831
Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp Ile Leu Arg His Pro
230 235 240 245
ggc ttc acc atg atg gca gca atc ctg gca tac acc ata gga acg aca 879
Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr Thr Ile Gly Thr Thr
250 255 260
cat ttc caa aga gcc ctg att ttc atc tta ctg aca gct gtc act cct 927
His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu Thr Ala Val Thr Pro
265 270 275
tca atg aca atg cgt tgc ata gga atg tca aat aga gac ttt gtg gaa 975
Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn Arg Asp Phe Val Glu
280 285 290
ggg gtt tca gga gga agc tgg gtt gac ata gtc tta gaa cat gga agc 1023
Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val Leu Glu His Gly Ser
295 300 305
tgt gtg acg acg atg gca aaa aac aaa cca aca ttg gat ttt gaa ctg 1071
Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu
310 315 320 325
ata aaa aca gaa gcc aaa cag cct gcc acc cta agg aag tac tgt ata 1119
Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cys Ile
330 335 340
gag gca aag cta acc aac aca aca aca gaa tct cgc tgc cca aca caa 1167
Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser Arg Cys Pro Thr Gln
345 350 355
ggg gaa ccc agc cta aat gaa gag cag gac aaa agg ttc gtc tgc aaa 1215
Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe Val Cys Lys
360 365 370
cac tcc atg gta gac aga gga tgg gga aat gga tgt gga cta ttt gga 1263
His Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly
375 380 385
aag gga ggc att gtg acc tgt gct atg ttc aga tgc aaa aag aac atg 1311
Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg Cys Lys Lys Asn Met
390 395 400 405
gaa gga aaa gtt gtg caa cca gaa aac ttg gaa tac acc att gtg ata 1359
Glu Gly Lys Val Val Gln Pro Glu Asn Leu Glu Tyr Thr Ile Val Ile
410 415 420
aca cct cac tca ggg gaa gag cat gca gtc gga aat gac aca gga aaa 1407
Thr Pro His Ser Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys
425 430 435
cat ggc aag gaa atc aaa ata aca cca cag agt tcc atc aca gaa gca 1455
His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile Thr Glu Ala
440 445 450
gaa ttg aca ggt tat ggc act gtc aca atg gag tgc tct cca aga acg 1503
Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr
455 460 465
ggc ctc gac ttc aat gag atg gtg ttg ctg cag atg gaa aat aaa gct 1551
Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln Met Glu Asn Lys Ala
470 475 480 485
tgg ctg gtg cac agg caa tgg ttc cta gac ctg ccg tta cca tgg ttg 1599
Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu
490 495 500
ccc gga gcg gac aca caa gag tca aat tgg ata cag aag gag aca ttg 1647
Pro Gly Ala Asp Thr Gln Glu Ser Asn Trp Ile Gln Lys Glu Thr Leu
505 510 515
gtc act ttc aaa aat ccc cat gcg aag aaa cag gat gtt gtt gtt tta 1695
Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln Asp Val Val Val Leu
520 525 530
gga tcc caa gaa ggg gcc atg cac aca gca ctt aca ggg gcc aca gaa 1743
Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu
535 540 545
atc caa atg tca tca gga aac tta ctc ttc aca gga cat ctc aag tgc 1791
Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys
550 555 560 565
agg ctg aga atg gac aag cta cag ctc aaa gga atg tca tac tct atg 1839
Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met
570 575 580
tgc aca gga aag ttt aaa gtt gtg aag gaa ata gca gaa aca caa cat 1887
Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gln His
585 590 595
gga aca ata gtt atc aga gtg caa tat gaa ggg gac ggc tct cca tgc 1935
Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys
600 605 610
aag atc cct ttt gag ata atg gat ttg gaa aaa aga cat gtc tta ggt 1983
Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly
615 620 625
cgc ctg att aca gtc aac cca att gtg aca gaa aaa gat agc cca gtc 2031
Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val
630 635 640 645
aac ata gaa gca gaa cct cca ttt gga gac agc tac atc atc ata gga 2079
Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly
650 655 660
gta gag ccg gga caa ctg aag ctc aac tgg ttt aag aaa gga agt tct 2127
Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser
665 670 675
atc ggc caa atg ttt gag aca aca atg agg ggg gcg aag aga atg gcc 2175
Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala
680 685 690
att tta ggt gac aca gcc tgg gat ttt gga tcc ttg gga gga gtg ttt 2223
Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly Val Phe
695 700 705
aca tct ata gga aag gct ctc cac caa gtc ttt gga gca atc tat gga 2271
Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe Gly Ala Ile Tyr Gly
710 715 720 725
gct gcc ttc agt ggg gtt tca tgg act atg aaa atc ctc ata gga gtc 2319
Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys Ile Leu Ile Gly Val
730 735 740
att atc aca tgg ata gga atg aat tca cgc agc acc tca ctg tct gtg 2367
Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser Thr Ser Leu Ser Val
745 750 755
aca cta gta ttg gtg gga att gtg aca ctg tat ttg gga gtc atg gtg 2415
Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr Leu Gly Val Met Val
760 765 770
cag gcc gat agt ggt tgc gtt gtg agc tgg aaa aac aaa gaa ctg aaa 2463
Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys
775 780 785
tgt ggc agt ggg att ttc atc aca gac aac gtg cac aca tgg aca gaa 2511
Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp Thr Glu
790 795 800 805
caa tac aaa ttc caa cca gaa tcc cct tca aaa cta gct tca gct atc 2559
Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala Ile
810 815 820
cag aaa gcc cat gaa gag gac att tgt gga atc cgc tca gta aca aga 2607
Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile Arg Ser Val Thr Arg
825 830 835
ctg gag aat ctg atg tgg aaa caa ata aca cca gaa ttg aat cac att 2655
Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn His Ile
840 845 850
cta tca gaa aat gag gtg aag tta act att atg aca gga gac atc aaa 2703
Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp Ile Lys
855 860 865
gga atc atg cag gca gga aaa cga tct ctg cgg cct cag ccc act gag 2751
Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr Glu
870 875 880 885
ctg aag tat tca tgg aaa aca tgg ggc aaa gca aaa atg ctc tct aca 2799
Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr
890 895 900
gag tct cat aac cag acc ttt ctc att gat ggc ccc gaa aca gca gaa 2847
Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr Ala Glu
905 910 915
tgc ccc aac aca aat aga gct tgg aat tcg ttg gaa gtt gaa gac tat 2895
Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr
920 925 930
ggc ttt gga gta ttc acc acc aat ata tgg cta aaa ttg aaa gaa aaa 2943
Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Lys Glu Lys
935 940 945
cag gat gta ttc tgc gac tca aaa ctc atg tca gcg gcc ata aaa gac 2991
Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile Lys Asp
950 955 960 965
aac aga gcc gtc cat gcc gat atg ggt tat tgg ata gaa agt gca ctc 3039
Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ala Leu
970 975 980
aat gac aca tgg aag ata gag aaa gcc tct ttc att gaa gtt aaa aac 3087
Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val Lys Asn
985 990 995
tgc cac tgg cca aaa tca cac acc ctc tgg agc aat gga gtg cta gaa 3135
Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu
1000 1005 1010
agt gag atg ata att cca aag aat ctc gct gga cca gtg tct caa cac 3183
Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly Pro Val Ser Gln His
1015 1020 1025
aac tat aga cca ggc tac cat aca caa ata aca gga cca tgg cat cta 3231
Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr Gly Pro Trp His Leu
1030 1035 1040 1045
ggt aag ctt gag atg gac ttt gat ttc tgt gat gga aca aca gtg gta 3279
Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val
1050 1055 1060
gtg act gag gac tgc gga aat aga gga ccc tct ttg aga aca acc act 3327
Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr
1065 1070 1075
gcc tct gga aaa ctc ata aca gaa tgg tgc tgc cga tct tgc aca tta 3375
Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu
1080 1085 1090
cca ccg cta aga tac aga ggt gag gat ggg tgc tgg tac ggg atg gaa 3423
Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu
1095 1100 1105
atc aga cca ttg aag gag aaa gaa gag aat ttg gtc aac tcc ttg gtc 3471
Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val
1110 1115 1120 1125
aca gct gga cat ggg cag gtc gac aac ttt tca cta gga gtc ttg gga 3519
Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly
1130 1135 1140
atg gca ttg ttc ctg gag gaa atg ctt agg acc cga gta gga acg aaa 3567
Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys
1145 1150 1155
cat gca ata cta cta gtt gca gtt tct ttt gtg aca ttg atc aca ggg 3615
His Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
aac atg tcc ttt aga gac ctg gga aga gtg atg gtt atg gta ggc gcc 3663
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly Ala
1175 1180 1185
act atg acg gat gac ata ggt atg ggc gtg act tat ctt gcc cta cta 3711
Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala Leu Leu
1190 1195 1200 1205
gca gcc ttc aaa gtc aga cca act ttt gca gct gga cta ctc ttg aga 3759
Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu Arg
1210 1215 1220
aag ctg acc tcc aag gaa ttg atg atg act act ata gga att gta ctc 3807
Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile Gly Ile Val Leu
1225 1230 1235
ctc tcc cag agc acc ata cca gag acc att ctt gag ttg act gat gcg 3855
Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu Glu Leu Thr Asp Ala
1240 1245 1250
tta gcc tta ggc atg atg gtc ctc aaa atg gtg aga aat atg gaa aag 3903
Leu Ala Leu Gly Met Met Val Leu Lys Met Val Arg Asn Met Glu Lys
1255 1260 1265
tat caa ttg gca gtg act atc atg gct atc ttg tgc gtc cca aac gca 3951
Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu Cys Val Pro Asn Ala
1270 1275 1280 1285
gtg ata tta caa aac gca tgg aaa gtg agt tgc aca ata ttg gca gtg 3999
Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys Thr Ile Leu Ala Val
1290 1295 1300
gtg tcc gtt tcc cca ctg ttc tta aca tcc tca cag caa aaa aca gat 4047
Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser Gln Gln Lys Thr Asp
1305 1310 1315
tgg ata cca tta gca ttg acg atc aaa ggt ctc aat cca aca gct att 4095
Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile
1320 1325 1330
ttt cta aca acc ctc tca aga acc agc aag aaa agg agc tgg cca tta 4143
Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu
1335 1340 1345
aat gag gct atc atg gca gtc ggg atg gtg agc att tta gcc agt tct 4191
Asn Glu Ala Ile Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser
1350 1355 1360 1365
ctc cta aaa aat gat att ccc atg aca gga cca tta gtg gct gga ggg 4239
Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly
1370 1375 1380
ctc ctc act gtg tgc tac gtg ctc act gga cga tcg gcc gat ttg gaa 4287
Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu
1385 1390 1395
ctg gag aga gca gcc gat gtc aaa tgg gaa gac cag gca gag ata tca 4335
Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
gga agc agt cca atc ctg tca ata aca ata tca gaa gat ggt agc atg 4383
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser Met
1415 1420 1425
tcg ata aaa aat gaa gag gaa gaa caa aca ctg acc ata ctc att aga 4431
Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu Ile Arg
1430 1435 1440 1445
aca gga ttg ctg gtg atc tca gga ctt ttt cct gta tca ata cca atc 4479
Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser Ile Pro Ile
1450 1455 1460
acg gca gca gca tgg tac ctg tgg gaa gtg aag aaa caa cgg gcc gga 4527
Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gln Arg Ala Gly
1465 1470 1475
gta ttg tgg gat gtt cct tca ccc cca ccc atg gga aag gct gaa ctg 4575
Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys Ala Glu Leu
1480 1485 1490
gaa gat gga gcc tat aga att aag caa aaa ggg att ctt gga tat tcc 4623
Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile Leu Gly Tyr Ser
1495 1500 1505
cag atc gga gcc gga gtt tac aaa gaa gga aca ttc cat aca atg tgg 4671
Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His Thr Met Trp
1510 1515 1520 1525
cat gtc aca cgt ggc gct gtt cta atg cat aaa gga aag agg att gaa 4719
His Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys Arg Ile Glu
1530 1535 1540
cca aca tgg gcg gac gtc aag aaa gac cta ata tca tat gga gga ggc 4767
Pro Thr Trp Ala Asp Val Lys Lys Asp Leu Ile Ser Tyr Gly Gly Gly
1545 1550 1555
tgg aag tta gaa gga gaa tgg aag gaa gga gaa gaa gtc cag gta ttg 4815
Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu Glu Val Gln Val Leu
1560 1565 1570
gca ctg gag cct gga aaa aat cca aga gcc gtc caa acg aaa cct ggt 4863
Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly
1575 1580 1585
ctt ttc aaa acc aac gcc gga aca ata ggt gct gta tct ctg gac ttt 4911
Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe
1590 1595 1600 1605
tct cct gga acg tca gga tct cca att atc gac aaa aaa gga aaa gtt 4959
Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val
1610 1615 1620
gtg ggt ctt tat ggt aat ggt gtt gtt aca agg agt gga gca tat gtg 5007
Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val
1625 1630 1635
agt gct ata gcc cag act gaa aaa agc att gaa gac aac cca gag atc 5055
Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
gaa gat cac att ttc cga aag aga aga ctg acc atc atg gac ctc cac 5103
Glu Asp His Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu His
1655 1660 1665
cca gga gcg gga aag acg aag aga tac ctt ccg gcc ata gtc aga gaa 5151
Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val Arg Glu
1670 1675 1680 1685
gct ata aaa cgg ggt ttg aga aca tta atc ttg gcc ccc act aga gtt 5199
Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro Thr Arg Val
1690 1695 1700
gtg gca gct gaa atg gag gaa gcc ctt aga gga ctt cca ata aga tac 5247
Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile Arg Tyr
1705 1710 1715
cag acc cca gcc atc aga gct gag cac acc ggg cgg gag att gtg gac 5295
Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly Arg Glu Ile Val Asp
1720 1725 1730
cta atg tgt cat gcc aca ttt acc atg agg ctg cta tca cca gtt aga 5343
Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser Pro Val Arg
1735 1740 1745
gtg cca aac tac aac ctg att atc atg gac gaa gcc cat ttc aca gac 5391
Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala His Phe Thr Asp
1750 1755 1760 1765
cca gca agt ata gca gct aga gga tac atc tca act cga gtg gag atg 5439
Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg Val Glu Met
1770 1775 1780
ggt gag gca gct ggg att ttt atg aca gcc act ccc ccg gga agc aga 5487
Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr Pro Pro Gly Ser Arg
1785 1790 1795
gac cca ttt cct cag agc aat gca cca atc ata gat gaa gaa aga gaa 5535
Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu
1800 1805 1810
atc cct gaa cgc tcg tgg aat tcc gga cat gaa tgg gtc acg gat ttt 5583
Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe
1815 1820 1825
aaa ggg aag act gtt tgg ttc gtt cca agt ata aaa gca gga aat gat 5631
Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp
1830 1835 1840 1845
ata gca gct tgc ctg agg aaa aat gga aag aaa gtg ata caa ctc agt 5679
Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser
1850 1855 1860
agg aag acc ttt gat tct gag tat gtc aag act aga acc aat gat tgg 5727
Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp
1865 1870 1875
gac ttc gtg gtt aca act gac att tca gaa atg ggt gcc aat ttc aag 5775
Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
gct gag agg gtt ata gac ccc aga cgc tgc atg aaa cca gtc ata cta 5823
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile Leu
1895 1900 1905
aca gat ggt gaa gag cgg gtg att ctg gca gga cct atg cca gtg acc 5871
Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val Thr
1910 1915 1920 1925
cac tct agt gca gca caa aga aga ggg aga ata gga aga aat cca aaa 5919
His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Lys
1930 1935 1940
aat gag aat gac cag tac ata tac atg ggg gaa cct ctg gaa aat gat 5967
Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu Glu Asn Asp
1945 1950 1955
gaa gac tgt gca cac tgg aaa gaa gct aaa atg ctc cta gat aac atc 6015
Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu Asp Asn Ile
1960 1965 1970
aac acg cca gaa gga atc att cct agc atg ttc gaa cca gag cgt gaa 6063
Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu Pro Glu Arg Glu
1975 1980 1985
aag gtg gat gcc att gat ggc gaa tac cgc ttg aga gga gaa gca agg 6111
Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu Arg Gly Glu Ala Arg
1990 1995 2000 2005
aaa acc ttt gta gac tta atg aga aga gga gac cta cca gtc tgg ttg 6159
Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro Val Trp Leu
2010 2015 2020
gcc tac aga gtg gca gct gaa ggc atc aac tac gca gac aga agg tgg 6207
Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr Ala Asp Arg Arg Trp
2025 2030 2035
tgt ttt gat gga gtc aag aac aac caa atc cta gaa gaa aac gtg gaa 6255
Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu Glu Glu Asn Val Glu
2040 2045 2050
gtt gaa atc tgg aca aaa gaa ggg gaa agg aag aaa ttg aaa ccc aga 6303
Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg
2055 2060 2065
tgg ttg gat gct agg atc tat tct gac cca ctg gcg cta aaa gaa ttt 6351
Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe
2070 2075 2080 2085
aag gaa ttt gca gcc gga aga aag tct ctg acc ctg aac cta atc aca 6399
Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr
2090 2095 2100
gaa atg ggt agg ctc cca acc ttc atg act cag aag gca aga gac gca 6447
Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala
2105 2110 2115
ctg gac aac tta gca gtg ctg cac acg gct gag gca ggt gga agg gcg 6495
Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
tac aac cat gct ctc agt gaa ctg ccg gag acc ctg gag aca ttg ctt 6543
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu
2135 2140 2145
tta ctg aca ctt ctg gct aca gtc acg gga ggg atc ttt tta ttc ttg 6591
Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu Phe Leu
2150 2155 2160 2165
atg agc gca agg ggc ata ggg aag atg acc ctg gga atg tgc tgc ata 6639
Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met Cys Cys Ile
2170 2175 2180
atc acg gct agc atc ctc cta tgg tac gca caa ata cag cca cac tgg 6687
Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile Gln Pro His Trp
2185 2190 2195
ata gca gct tca ata ata ctg gag ttt ttt ctc ata gtt ttg ctt att 6735
Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Ile Val Leu Leu Ile
2200 2205 2210
cca gaa cct gaa aaa cag aga aca ccc caa gac aac caa ctg acc tac 6783
Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn Gln Leu Thr Tyr
2215 2220 2225
gtt gtc ata gcc atc ctc aca gtg gtg gcc gca acc atg gca aac gag 6831
Val Val Ile Ala Ile Leu Thr Val Val Ala Ala Thr Met Ala Asn Glu
2230 2235 2240 2245
atg ggt ttc cta gaa aaa acg aag aaa gat ctc gga ttg gga agc att 6879
Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu Gly Leu Gly Ser Ile
2250 2255 2260
gca acc cag caa ccc gag agc aac atc ctg gac ata gat cta cgt cct 6927
Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp Ile Asp Leu Arg Pro
2265 2270 2275
gca tca gca tgg acg ctg tat gcc gtg gcc aca aca ttt gtt aca cca 6975
Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Phe Val Thr Pro
2280 2285 2290
atg ttg aga cat agc att gaa aat tcc tca gtg aat gtg tcc cta aca 7023
Met Leu Arg His Ser Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr
2295 2300 2305
gct ata gcc aac caa gcc aca gtg tta atg ggt ctc ggg aaa gga tgg 7071
Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp
2310 2315 2320 2325
cca ttg tca aag atg gac atc gga gtt ccc ctt ctc gcc att gga tgc 7119
Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys
2330 2335 2340
tac tca caa gtc aac ccc ata act ctc aca gca gct ctt ttc tta ttg 7167
Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu
2345 2350 2355
gta gca cat tat gcc atc ata ggg cca gga ctc caa gca aaa gca acc 7215
Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
aga gaa gct cag aaa aga gca gcg gcg ggc atc atg aaa aac cca act 7263
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro Thr
2375 2380 2385
gtc gat gga ata aca gtg att gac cta gat cca ata cct tat gat cca 7311
Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr Asp Pro
2390 2395 2400 2405
aag ttt gaa aag cag ttg gga caa gta atg ctc cta gtc ctc tgc gtg 7359
Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu Cys Val
2410 2415 2420
act caa gta ttg atg atg agg act aca tgg gct ctg tgt gag gct tta 7407
Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu Ala Leu
2425 2430 2435
acc tta gct acc ggg ccc atc tcc aca ttg tgg gaa gga aat cca ggg 7455
Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp Glu Gly Asn Pro Gly
2440 2445 2450
agg ttt tgg aac act acc att gcg gtg tca atg gct aac att ttt aga 7503
Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala Asn Ile Phe Arg
2455 2460 2465
ggg agt tac ttg gcc gga gct gga ctt ctc ttt tct att atg aag aac 7551
Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe Ser Ile Met Lys Asn
2470 2475 2480 2485
aca acc aac aca aga agg gga act ggc aac ata gga gag acg ctt gga 7599
Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile Gly Glu Thr Leu Gly
2490 2495 2500
gag aaa tgg aaa agc cga ttg aac gca ttg gga aaa agt gaa ttc cag 7647
Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly Lys Ser Glu Phe Gln
2505 2510 2515
atc tac aag aaa agt gga atc cag gaa gtg gat aga acc tta gca aaa 7695
Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp Arg Thr Leu Ala Lys
2520 2525 2530
gaa ggc att aaa aga gga gaa acg gac cat cac gct gtg tcg cga ggc 7743
Glu Gly Ile Lys Arg Gly Glu Thr Asp His His Ala Val Ser Arg Gly
2535 2540 2545
tca gca aaa ctg aga tgg ttc gtt gag aga aac atg gtc aca cca gaa 7791
Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu
2550 2555 2560 2565
ggg aaa gta gtg gac ctc ggt tgt ggc aga gga ggc tgg tca tac tat 7839
Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr
2570 2575 2580
tgt gga gga cta aag aat gta aga gaa gtc aaa ggc cta aca aaa gga 7887
Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly
2585 2590 2595
gga cca gga cac gaa gaa ccc atc ccc atg tca aca tat ggg tgg aat 7935
Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
cta gtg cgt ctt caa agt gga gtt gac gtt ttc ttc atc ccg cca gaa 7983
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro Glu
2615 2620 2625
aag tgt gac aca tta ttg tgt gac ata ggg gag tca tca cca aat ccc 8031
Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro
2630 2635 2640 2645
aca gtg gaa gca gga cga aca ctc aga gtc ctt aac tta gta gaa aat 8079
Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu Asn
2650 2655 2660
tgg ttg aac aac aac act caa ttt tgc ata aag gtt ctc aac cca tat 8127
Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val Leu Asn Pro Tyr
2665 2670 2675
atg ccc tca gtc ata gaa aaa atg gaa gca cta caa agg aaa tat gga 8175
Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu Gln Arg Lys Tyr Gly
2680 2685 2690
gga gcc tta gtg agg aat cca ctc tca cga aac tcc aca cat gag atg 8223
Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met
2695 2700 2705
tac tgg gta tcc aat gct tcc ggg aac ata gtg tca tca gtg aac atg 8271
Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val Ser Ser Val Asn Met
2710 2715 2720 2725
att tca agg atg ttg atc aac aga ttt aca atg aga tac aag aaa gcc 8319
Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met Arg Tyr Lys Lys Ala
2730 2735 2740
act tac gag ccg gat gtt gac ctc gga agc gga acc cgt aac atc ggg 8367
Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly Thr Arg Asn Ile Gly
2745 2750 2755
att gaa agt gag ata cca aac cta gat ata att ggg aaa aga ata gaa 8415
Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu
2760 2765 2770
aaa ata aag caa gag cat gaa aca tca tgg cac tat gac caa gac cac 8463
Lys Ile Lys Gln Glu His Glu Thr Ser Trp His Tyr Asp Gln Asp His
2775 2780 2785
cca tac aaa acg tgg gca tac cat ggt agc tat gaa aca aaa cag act 8511
Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr
2790 2795 2800 2805
gga tca gca tca tcc atg gtc aac gga gtg gtc agg ctg ctg aca aaa 8559
Gly Ser Ala Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys
2810 2815 2820
cct tgg gac gtt gtc ccc atg gtg aca cag atg gca atg aca gac acg 8607
Pro Trp Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr
2825 2830 2835
act cca ttt gga caa cag cgc gtt ttt aaa gag aaa gtg gac acg aga 8655
Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
acc caa gaa ccg aaa gaa ggc acg aag aaa cta atg aaa ata aca gca 8703
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala
2855 2860 2865
gag tgg ctt tgg aaa gaa tta ggg aag aaa aag aca ccc agg atg tgc 8751
Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys
2870 2875 2880 2885
acc aga gaa gaa ttc aca aga aag gtg aga agc aat gca gcc ttg ggg 8799
Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly
2890 2895 2900
gcc ata ttc act gat gag aac aag tgg aag tcg gca cgt gag gct gtt 8847
Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val
2905 2910 2915
gaa gat agt agg ttt tgg gag ctg gtt gac aag gaa agg aat ctc cat 8895
Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His
2920 2925 2930
ctt gaa gga aag tgt gaa aca tgt gtg tac aac atg atg gga aaa aga 8943
Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met Met Gly Lys Arg
2935 2940 2945
gag aag aag cta ggg gaa ttc ggc aag gca aaa ggc agc aga gcc ata 8991
Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile
2950 2955 2960 2965
tgg tac atg tgg ctt gga gca cgc ttc tta gag ttt gaa gcc cta gga 9039
Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly
2970 2975 2980
ttc tta aat gaa gat cac tgg ttc tcc aga gag aac tcc ctg agt gga 9087
Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly
2985 2990 2995
gtg gaa gga gaa ggg ctg cac aag cta ggt tac att cta aga gac gtg 9135
Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr Ile Leu Arg Asp Val
3000 3005 3010
agc aag aaa gag gga gga gca atg tat gcc gat gac acc gca gga tgg 9183
Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp
3015 3020 3025
gat aca aaa atc aca cta gaa gac cta aaa aat gaa gag atg gta aca 9231
Asp Thr Lys Ile Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr
3030 3035 3040 3045
aac cac atg gaa gga gaa cac aag aaa cta gcc gag gcc att ttc aaa 9279
Asn His Met Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys
3050 3055 3060
cta acg tac caa aac aag gtg gtg cgt gtg caa aga cca aca cca aga 9327
Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg
3065 3070 3075
ggc aca gta atg gac atc ata tcg aga aga gac caa aga ggt agt gga 9375
Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
caa gtt ggc acc tat gga ctc aat act ttc acc aat atg gaa gcc caa 9423
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln
3095 3100 3105
cta atc aga cag atg gag gga gaa gga gtc ttt aaa agc att cag cac 9471
Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His
3110 3115 3120 3125
cta aca atc aca gaa gaa atc gct gtg caa aac tgg tta gca aga gtg 9519
Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu Ala Arg Val
3130 3135 3140
ggg cgc gaa agg tta tca aga atg gcc atc agt gga gat gat tgt gtt 9567
Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly Asp Asp Cys Val
3145 3150 3155
gtg aaa cct tta gat gac agg ttc gca agc gct tta aca gct cta aat 9615
Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn
3160 3165 3170
gac atg gga aag att agg aaa gac ata caa caa tgg gaa cct tca aga 9663
Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln Trp Glu Pro Ser Arg
3175 3180 3185
gga tgg aat gat tgg aca caa gtg ccc ttc tgt tca cac cat ttc cat 9711
Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys Ser His His Phe His
3190 3195 3200 3205
gag tta atc atg aaa gac ggt cgc gta ctc gtt gtt cca tgt aga aac 9759
Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val Val Pro Cys Arg Asn
3210 3215 3220
caa gat gaa ctg att ggc aga gcc cga atc tcc caa gga gca ggg tgg 9807
Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp
3225 3230 3235
tct ttg cgg gag acg gcc tgt ttg ggg aag tct tac gcc caa atg tgg 9855
Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp
3240 3245 3250
agc ttg atg tac ttc cac aga cgc gac ctc agg ctg gcg gca aat gct 9903
Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala
3255 3260 3265
att tgc tcg gca gta cca tca cat tgg gtt cca aca agt cga aca acc 9951
Ile Cys Ser Ala Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr
3270 3275 3280 3285
tgg tcc ata cat gct aaa cat gaa tgg atg aca acg gaa gac atg ctg 9999
Trp Ser Ile His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu
3290 3295 3300
aca gtc tgg aac agg gtg tgg att caa gaa aac cca tgg atg gaa gac 10047
Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp
3305 3310 3315
aaa act cca gtg gaa aca tgg gag gaa atc cca tac ttg ggg aaa aga 10095
Lys Thr Pro Val Glu Thr Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
gaa gac caa tgg tgc ggc tca ttg att ggg tta aca agc agg gcc acc 10143
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr
3335 3340 3345
tgg gca aag aac atc caa gca gca ata aat caa gtt aga tcc ctt ata 10191
Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser Leu Ile
3350 3355 3360 3365
ggc aat gaa gaa tac aca gat tac atg cca tcc atg aaa aga ttc aga 10239
Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe Arg
3370 3375 3380
aga gaa gag gaa gaa gca gga gtt ctg tgg t agaaagcaaa actaacatga 10290
Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3385 3390
aacaaggcta gaagtcaggt cggattaagc catagtacgg aaaaaactat gctacctgtg 10350
agccccgtcc aaggacgtta aaagaagtca ggccatcata aatgccatag cttgagtaaa 10410
ctatgcagcc tgtagctcca cctgagaagg tgtaaaaaat ccgggaggcc acaaaccatg 10470
gaagctgtac gcatggcgta gtggactagc ggttagggga gacccctccc ttacaaatcg 10530
cagcaacaat gggggcccaa ggcgagatga agctgtagtc tcgctggaag gactagaggt 10590
tagaggagac ccccccgaaa caaaaaacag catattgacg ctgggaaaga ccagagatcc 10650
tgctgtctcc tcagcatcat tccaggcaca gaacgccaga aaatggaatg gtgctgttga 10710
atcaacaggt tct 10723
<210> 2
<211> 3391
<212> PRT
<213> Dengue virus
<400> 2
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val
115 120 125
Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly
130 135 140
Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu
145 150 155 160
Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala
195 200 205
Leu Val Pro His Val Arg Met Gly Leu Glu Thr Arg Thr Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp
225 230 235 240
Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr
245 250 255
Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu
260 265 270
Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn
275 280 285
Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val
290 295 300
Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu
325 330 335
Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys
355 360 365
Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg
385 390 395 400
Cys Lys Lys Asn Met Glu Gly Lys Val Val Gln Pro Glu Asn Leu Glu
405 410 415
Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly
420 425 430
Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser
435 440 445
Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu
450 455 460
Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln
465 470 475 480
Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Glu Ser Asn Trp Ile
500 505 510
Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln
515 520 525
Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu
530 535 540
Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr
545 550 555 560
Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly
565 570 575
Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile
580 585 590
Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly
595 600 605
Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys
610 615 620
Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu
625 630 635 640
Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser
645 650 655
Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe
660 665 670
Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly
675 680 685
Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
690 695 700
Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe
705 710 715 720
Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys
725 730 735
Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser
740 745 750
Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly
1010 1015 1020
Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr
1025 1030 1035 1040
Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp
1045 1050 1055
Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser
1060 1065 1070
Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys
1075 1080 1085
Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys
1090 1095 1100
Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu
1105 1110 1115 1120
Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser
1125 1130 1135
Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr
1140 1145 1150
Arg Val Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val
1155 1160 1165
Thr Leu Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met
1170 1175 1180
Val Met Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr
1185 1190 1195 1200
Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala
1205 1210 1215
Gly Leu Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr
1220 1225 1230
Ile Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val
1250 1255 1260
Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu
1265 1270 1275 1280
Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys
1285 1290 1295
Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser
1300 1305 1310
Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu
1315 1320 1325
Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys
1330 1335 1340
Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser
1345 1350 1355 1360
Ile Leu Ala Ser Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro
1365 1370 1375
Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg
1380 1385 1390
Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp
1395 1400 1405
Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser
1410 1415 1420
Glu Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu
1425 1430 1435 1440
Thr Ile Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro
1445 1450 1455
Val Ser Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys
1460 1465 1470
Lys Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly
1490 1495 1500
Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr
1505 1510 1515 1520
Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys
1525 1530 1535
Gly Lys Arg Ile Glu Pro Thr Trp Ala Asp Val Lys Lys Asp Leu Ile
1540 1545 1550
Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu
1555 1560 1565
Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val
1570 1575 1580
Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala
1585 1590 1595 1600
Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp
1605 1610 1615
Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg
1620 1625 1630
Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu
1635 1640 1645
Asp Asn Pro Glu Ile Glu Asp His Ile Phe Arg Lys Arg Arg Leu Thr
1650 1655 1660
Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro
1665 1670 1675 1680
Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu
1685 1690 1695
Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly
1700 1705 1710
Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu
1730 1735 1740
Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu
1745 1750 1755 1760
Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser
1765 1770 1775
Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr
1780 1785 1790
Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile
1795 1800 1805
Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu
1810 1815 1820
Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile
1825 1830 1835 1840
Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys
1845 1850 1855
Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr
1860 1865 1870
Arg Thr Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met
1875 1880 1885
Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met
1890 1895 1900
Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly
1905 1910 1915 1920
Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile
1925 1930 1935
Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu
1940 1945 1950
Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe
1970 1975 1980
Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu
1985 1990 1995 2000
Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp
2005 2010 2015
Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr
2020 2025 2030
Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu
2035 2040 2045
Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys
2050 2055 2060
Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu
2065 2070 2075 2080
Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr
2085 2090 2095
Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln
2100 2105 2110
Lys Ala Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu
2115 2120 2125
Ala Gly Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr
2130 2135 2140
Leu Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly
2145 2150 2155 2160
Ile Phe Leu Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu
2165 2170 2175
Gly Met Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln
2180 2185 2190
Ile Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp
2210 2215 2220
Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala
2225 2230 2235 2240
Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu
2245 2250 2255
Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp
2260 2265 2270
Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr
2275 2280 2285
Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val
2290 2295 2300
Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly
2305 2310 2315 2320
Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu
2325 2330 2335
Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala
2340 2345 2350
Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu
2355 2360 2365
Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile
2370 2375 2380
Met Lys Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro
2385 2390 2395 2400
Ile Pro Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu
2405 2410 2415
Leu Val Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala
2420 2425 2430
Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met
2450 2455 2460
Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe
2465 2470 2475 2480
Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile
2485 2490 2495
Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly
2500 2505 2510
Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp
2515 2520 2525
Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His
2530 2535 2540
Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn
2545 2550 2555 2560
Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly
2565 2570 2575
Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys
2580 2585 2590
Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser
2595 2600 2605
Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe
2610 2615 2620
Phe Ile Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu
2625 2630 2635 2640
Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu
2645 2650 2655
Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys
2660 2665 2670
Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn
2690 2695 2700
Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val
2705 2710 2715 2720
Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met
2725 2730 2735
Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly
2740 2745 2750
Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile
2755 2760 2765
Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His
2770 2775 2780
Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr
2785 2790 2795 2800
Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser Met Val Asn Gly Val Val
2805 2810 2815
Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met
2820 2825 2830
Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu
2835 2840 2845
Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu
2850 2855 2860
Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys
2865 2870 2875 2880
Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser
2885 2890 2895
Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser
2900 2905 2910
Ala Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn
2930 2935 2940
Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys
2945 2950 2955 2960
Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu
2965 2970 2975
Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu
2980 2985 2990
Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr
2995 3000 3005
Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp
3010 3015 3020
Asp Thr Ala Gly Trp Asp Thr Lys Ile Thr Leu Glu Asp Leu Lys Asn
3025 3030 3035 3040
Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Leu Ala
3045 3050 3055
Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln
3060 3065 3070
Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp
3075 3080 3085
Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr
3090 3095 3100
Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe
3105 3110 3115 3120
Lys Ser Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn
3125 3130 3135
Trp Leu Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser
3140 3145 3150
Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln
3170 3175 3180
Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys
3185 3190 3195 3200
Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val
3205 3210 3215
Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser
3220 3225 3230
Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser
3235 3240 3245
Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg
3250 3255 3260
Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro
3265 3270 3275 3280
Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr
3285 3290 3295
Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn
3300 3305 3310
Pro Trp Met Glu Asp Lys Thr Pro Val Glu Thr Trp Glu Glu Ile Pro
3315 3320 3325
Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu
3330 3335 3340
Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln
3345 3350 3355 3360
Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser
3365 3370 3375
Met Lys Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 3
<211> 10723
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10723)
<223> Wild-type DEN2 strain 16681
<400> 3
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480
agcagacaag agaaagggaa aagtcttctg tttaaaacag aggatggcgt gaacatgtgt 540
accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600
cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660
gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720
ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780
ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840
atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900
ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960
agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020
agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080
gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140
acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200
aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260
ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320
gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380
catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440
tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500
acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560
cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620
tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680
gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740
gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800
atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860
gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920
gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980
ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040
gcagaacctc cattcggaga cagctacatc atcataggag tagagccggg acaactgaag 2100
ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160
gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220
tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280
agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340
aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagagggc 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgctc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctga gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgttcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgga 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 4
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 4
gttttcccag tcacgacacg tggaccgaca aagacag 37
<210> 5
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
aacagctatg accatgttcc tcctgaaacc ccttcc 36
<210> 6
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
gttttcccag tcacgacatc acgtacaagt gtcccc 36
<210> 7
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 7
aacagctatg accatgagca acaccatctc attgaag 37
<210> 8
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 8
gttttcccag tcacgactgc aaccagaaaa cttggaatac ac 42
<210> 9
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 9
aacagctatg accatggctc catagattgc tccaaagac 39
<210> 10
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 10
gttttcccag tcacgacccc agtcaacata gaagcagaac c 41
<210> 11
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 11
aacagctatg accatgccaa agccatagtc ttcaacttcc 40
<210> 12
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 12
gttttcccag tcacgacatc atgcaggcag gaaaac 36
<210> 13
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 13
aacagctatg accatgacca taaccatcac tcttccc 37
<210> 14
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 14
aacagctatg accatgacca taaccatcac tcttccc 37
<210> 15
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 15
aacagctatg accatggctc tctccagttc caaatc 36
<210> 16
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 16
gttttcccag tcacgacaag aaccagcaag aaaaggag 38
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 17
aacagctatg accatgcacc attaccataa agacccac 38
<210> 18
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 18
gttttcccag tcacgacttg aaccatcatg ggcggac 37
<210> 19
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 19
aacagctatg accatgtcct gcttttatac ttggaacgaa c 41
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 20
gttttcccag tcacgacaag cccatttcac agaccc 36
<210> 21
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 21
aacagctatg accatgtcaa tttcttcctt tccccttc 38
<210> 22
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 22
gttttcccag tcacgacgag aggagaagca aggaaaac 38
<210> 23
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 23
aacagctatg accatgaggg acacattcac tgagg 35
<210> 24
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 24
gttttcccag tcacgacaca gagaacaccc caagac 36
<210> 25
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 25
aacagctatg accatgtcca cttcctggat tccac 35
<210> 26
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 26
gttttcccag tcacgacaca agtaatgctc ctagtcctc 39
<210> 27
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 27
aacagctatg accatgttca ctgatgacac tatgttcc 38
<210> 28
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 28
gttttcccag tcacgacgtc atcaccaaat cccacag 37
<210> 29
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 29
aacagctatg accatggctt cttctctctt tttcccatc 39
<210> 30
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 30
gttttcccag tcacgacaag gtgagaagca atgcag 36
<210> 31
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 31
aacagctatg accatgtgga aatggtgtga acagaag 37
<210> 32
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 32
gttttcccag tcacgacgca ttcagcacct aacaatcac 39
<210> 33
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 33
aacagctatg accatgggca tttatgatgg cctga 35
<210> 34
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 34
ccatggaagc tgtacgc 17
<210> 35
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 35
aacagctatg accatgtgat tcaacagcac cattcc 36
<210> 36
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 36
gttttcccag tcacgac 17
<210> 37
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 37
aacagctatg accatg 16
<210> 38
<211> 10723
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10723)
<223> LAV2
<400> 38
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480
agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540
accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600
cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660
gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720
ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780
ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840
atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900
ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960
agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020
agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080
gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140
acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200
aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260
ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320
gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380
catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440
tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500
acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560
cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620
tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680
gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740
gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800
atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860
gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920
gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980
ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040
gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100
ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160
gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220
tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280
agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340
aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgw gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt tgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 39
<211> 10735
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10735)
<223> VDV1
<400> 39
agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60
ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120
tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180
ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240
tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300
gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360
aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420
gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480
caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540
cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600
gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660
gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720
ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780
tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840
gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900
cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960
agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020
ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080
ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140
caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200
ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260
aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320
agctcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380
ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440
tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500
agggctagat tttaacgaga tggtgttgct gacaatgaaa aagaaatcat ggcttgtcca 1560
caaacagtgg tttctagact taccactgcc ttggacctct ggggctttaa catcccaaga 1620
gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680
agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740
aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800
ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860
gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920
agacgcacca tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980
gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040
agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100
aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160
acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220
cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280
cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340
ttcaaggaac acgtcccttt cggtgatgtg catcgcagtt ggcatggtca cactgtacct 2400
aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460
atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520
ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580
gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640
attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagacgttag 2700
tggaatcttg gcccaaggaa aaaaaatgat taggccacaa cccatggaac acaaatactc 2760
gtggaaaagc tggggaaaag ctaaaatcat aggagcggat gtacagaaca ccaccttcat 2820
catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880
agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940
ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000
ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060
agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120
tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180
caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240
actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300
aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360
atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420
aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480
aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540
gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600
tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660
caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720
aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780
tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840
gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900
acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960
ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020
gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080
accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140
caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200
tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260
atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320
agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380
gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440
ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500
gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560
aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620
ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680
gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740
agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800
agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860
taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920
atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980
ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040
gcctctacca gagattgagg acaaggtgtt taggaaaaga aacttaacaa taatggacct 5100
acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160
aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220
ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280
aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340
gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400
catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460
tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520
agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580
ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640
ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700
gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760
gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820
actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880
tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940
ttacatggga cagcctttaa aaaatgatga ggaccacgct cattggacag aagcaaagat 6000
gctccttgac aatataaaca caccagaagg gattatccca gccctctttg agccggagag 6060
agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120
cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga 6180
aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240
ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300
tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360
tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420
acatttgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480
acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540
gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600
aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660
atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720
gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780
atatgtggtg ataggtctgt tattcgtgat attgacagtg gcagccaatg agatgggatt 6840
attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900
tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960
cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020
cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080
atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140
gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200
actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260
tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320
tgaaaaacag ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380
gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440
ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500
tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560
aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620
actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680
ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740
gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800
agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860
agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920
ggcgacctat ggatggaacc tagtaaggct gcactccgga aaagatgtat tttttatacc 7980
acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040
agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100
attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160
gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220
aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280
aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340
cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400
tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460
caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520
ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580
ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640
gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700
agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760
ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820
tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880
agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940
gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000
gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060
gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120
catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180
atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240
ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300
ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360
ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420
ccaactgata agacaaatgg agtctgaggg aatcttttta cccagcgaat tggaaacccc 9480
aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540
aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600
cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660
aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720
tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780
ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840
atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900
cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960
ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020
gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080
atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140
cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200
gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260
ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320
agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380
caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440
gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500
ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560
acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620
aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680
attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 10735
<210> 40
<211> 10735
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10735)
<223> LAV1/PDK13
<400> 40
agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60
ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120
tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180
ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240
tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300
gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360
aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420
gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480
caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540
cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600
gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660
gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720
ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780
tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840
gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900
cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960
agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020
ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080
ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140
caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200
ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260
aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320
agctcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380
ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440
tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500
agggctagat tttaacgaga tggtgttgct gacaatgaaa aagaaatcat ggcttgtcca 1560
caaacagtgg tttctagact taccactgcc ttggacctct ggggctttaa catcccaaga 1620
gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680
agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740
aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800
ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860
gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920
agacgcacca tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980
gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040
agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100
aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160
acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220
cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280
cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340
ttcaaggaac acgtcccttt cggtgatgtg catcgcagtt ggcatggtca cactgtacct 2400
aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460
atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520
ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580
gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640
attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagacgttag 2700
tggaatcttg gcccaaggga aaaaaatgat taggccacaa cccatggaac acaaatactc 2760
gtggaaaagc tggggaaaag ctaaaatcat aggagcggat gtacagaaca ccaccttcat 2820
catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880
agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940
ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000
ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060
agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120
tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180
caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240
actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300
aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360
atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420
aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480
aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540
gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600
tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660
caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720
aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780
tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840
gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900
acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960
ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020
gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080
accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140
caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200
tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260
atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320
agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380
gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440
ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500
gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560
aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620
ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680
gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740
agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800
agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860
taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920
atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980
ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040
gcctctacca gagattgagg acaaggtgtt taggaaaaga aacttaacaa taatggacct 5100
acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160
aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220
ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280
aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340
gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400
catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460
tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520
agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580
ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640
ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700
gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760
gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820
actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880
tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940
ttacatggga cagcctttaa acaatgatga ggaccacgct cattggacag aagcaaagat 6000
gctccttgac aatataaaca caccagaagg gattatccca gccctctttg agccggagag 6060
agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120
cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga 6180
aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240
ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300
tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360
tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420
acatttgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480
acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540
gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600
aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660
atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720
gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780
atatgtggtg ataggtctgt tattcgtgat attgacagtg gcagccaatg agatgggatt 6840
attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900
tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960
cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020
cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080
atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140
gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200
actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260
tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320
tgaaaaacag ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380
gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440
ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500
tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560
aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620
actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680
ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740
gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800
agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860
agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920
ggcgacctat ggatggaacc tagtaaagct gcactccgga aaagatgtat tttttatacc 7980
acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040
agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100
attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160
gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220
aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280
aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340
cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400
tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460
caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520
ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580
ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640
gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700
agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760
ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820
tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880
agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940
gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000
gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060
gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120
catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180
atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240
ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300
ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360
ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420
ccaactgata agacaaatgg agtctgaggg aatcttttta cccagcgaat tggaaacccc 9480
aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540
aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600
cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660
aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720
tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780
ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840
atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900
cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960
ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020
gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080
atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140
cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200
gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260
ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320
agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380
caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440
gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500
ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560
acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620
aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680
attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 10735
<210> 41
<211> 10699
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10699)
<223> LAV3
<400> 41
agttgttagt ctacgtggac cgacaagaac agtttcgact cggaagcttg cttaacgtag 60
tgctgacagt tttttattag agagcagatc tctgatgaac aaccaacgga aaaagacggg 120
aaaaccgtct atcaatatgc tgaaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180
ggcgaagaga ttctcaagag gattgctgaa cggccaagga ccaatgaaat tggttatggc 240
atttatagct ttcctcagat ttctagccat tccaccgaca gcaggagtct tggctagatg 300
gggtaccttt aagaagtcgg gggctattaa ggtcttaaaa ggcttcaaga aggagatctc 360
aaacatgctg agcattatca acaaacggaa aaagacatcg ctctgtctca tgatgatgtt 420
accagcaaca cttgctttcc acttaacttc acgagatgga gagccgcgca tgattgtggg 480
gaagaatgaa agaggaaaat ccctactttt caagacagcc tctggaatca acatgtgcac 540
actcatagct atggatctgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600
cattaccgaa gtggagcctg aagacattga ctgctggtgc aaccttacat cgacatgggt 660
gacttatgga acatgcaatc aagctggaga gcatagacgc gataagagat cagtggcgtt 720
agctccccat gttggcatgg gactggacac acgcactcaa acctggatgt cggctgaagg 780
agcttggaga caagtcgaga aggtagagac atgggccctt aggcacccag ggtttaccat 840
actagcccta tttcttgccc attacatagg cacttccttg acccagaaag tggttatttt 900
tatactatta atgctggtta ccccatccat gacaatgaga tgtgtaggag taggaaacag 960
agattttgtg gaaggcctat cgggagctac gtgggttgac gtggtgctcg agcacggtgg 1020
gtgtgtgact accatggcta agaacaagcc cacgctggac atagagcttc agaagaccga 1080
ggccacccaa ctggcgaccc taaggaagct atgcattgag ggaaaaatta ccaacataac 1140
aaccgactca agatgtccca cccaagggga agcgatttta cctgaggagc aggaccagaa 1200
ctacgtgtgt aagcatacat acgtggacag aggctgggga aacggttgtg gtttgtttgg 1260
caagggaagc ttggtgacat gcgcgaaatt tcaatgttta gaatcaatag agggaaaagt 1320
ggtgcaacat gagaacctca aatacaccgt catcatcaca gtgcacacag gagaccaaca 1380
ccaggtggga aatgaaacgc agggagtcac ggctgagata acaccccagg catcaaccgc 1440
tgaagccatt ttacctgaat atggaaccct cgggctagaa tgctcaccac ggacaggttt 1500
ggatttcaat gaaatgatct yattgacaat gaagaacaaa gcatggatgg tacatagaca 1560
atggttcttt gacttacccc taccatggac atcaggagct acagcagaaa caccaacttg 1620
gaacaggaaa gagcttcttg tgacatttaa aaatgcacat gcaaaaaagc aagaagtagt 1680
tgttcttgga tcacaagagg gagcaatgca tacagcactg acaggagcta cagagatcca 1740
aacctcagga ggcacaagta tctttgcggg gcacttaaaa tgtagactca agatggacaa 1800
attggaactc aaagggatga gctatgcaat gtgcttgggt agctttgtgt tgaagaaaga 1860
agtctccgaa acgcagcatg ggacaatact cattaaggtt gagtacaaag ggaaagatgc 1920
accctgcaag attcctttct ccacggagga tggacaagga aaagctcaca atggcagact 1980
gatcacagcc aatccagtgg tgaccaagaa ggaggagcct gtcaacattg aggctgaacc 2040
tccttttgga gaaagtaaca tagtaattgg aattggagac aaagccctga aaatcaactg 2100
gtacaagaag ggaagctcga ttgggaagat gttcgaggct actgccagag gtgcaaggcg 2160
catggccatc ttgggagaca cagcctggga ctttggatca gtgggtggtg ttttgaattc 2220
attagggaaa atggtccacc aaatatttgg gagtgcttac acagccctat ttggtggagt 2280
ctcctggatg atgaaaattg gaataggtgt cctcttaacc tggatagggt tgaactcaaa 2340
aaatacttct atgtcatttt catgcatcgc gataggaatc attacactct atctgggagc 2400
cgtggtgcaa gctgacatgg ggtgtgtcat aaactggaaa ggcaaagaac tcaaatgtgg 2460
aagtggaatt ttcgtcacta atgaggtcca cacctggaca gagcaataca aatttcaagc 2520
agactccccc aagagactgg caacagccat tgcaggcgct tgggaaaatg gagtgtgcgg 2580
aattaggtca acaaccagaa tggagaacct cttgtggaag caaatagcca atgaactgaa 2640
ttacatatta tgggaaaaca acattaaatt aacggtagtt gtaggcgaca taactggggt 2700
cttagagcaa gggaaaagaa cactaacacc acaacccatg gagctaaaat attcttggaa 2760
aacatgggga aaggcaaaaa tagtgacagc tgaaacacaa aattcctctt tcataataga 2820
tgggccaagc acaccggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880
ggattacggg ttcggagttt tcacaaccaa catatggctg aaactccgag aggtgtacac 2940
ccaactatgt gaccataggc taatgtcggc agccgtcaag gatgagaggg ctgtacatgc 3000
cgacatgggc tattggatag aaagccaaaa gaatgggagt tggaagctag aaaaagcatc 3060
cttcatagag gtgaaaacct gcacatggcc aaaatcacac actctctgga gcaatggtgt 3120
gctagagagt gacatgatta tcccaaagag tctagctggt cccatttcgc aacacaacca 3180
caggcccggg taccacaccc aaacggcagg accctggcac ttaggaaaat tggagctgga 3240
cttcaactat tgtgaaggaa caacagttgt catctcagaa aactgtggga caagaggccc 3300
atcattgaga acaacaacgg tgtcagggaa gttgatacac gaatggtgct gccgctcgtg 3360
cacacttcct cccctacgat acatgggaga agacggctgc tggtatggca tggaaatcag 3420
acccattaat gagaaagaag agaatatggt aaagtctcta gcctcagcag ggagtggaaa 3480
ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540
aggaaaattt gggaaaaaac acatgattgc aggggttctc ttcacgtttg tgctcctcct 3600
ctcagggcaa ataacatgga gagacatggc gcacacactc ataatgattg ggtccaacgc 3660
ctctgacaga atggggatgg gcgtcactta cctagctcta attgcaacat ttaaaattca 3720
gccactcctg gctttgggat tcttcctgag gaaactgaca tctagagaaa atttattgct 3780
gggagttggg ttggccatgg cagcaacgtt acgactgcca gaggacattg aacagatggc 3840
gaatggaatt gctttggggc tcatggctct taaactgata acacaatttg aaacatacca 3900
actatggacg gcattagttt ccctaacgtg ttcaaataca attttcacgt tgactgttgc 3960
ctggagaaca gccactctga ttttagccgg aatttcgctt ttgccagtgt gccagtcttc 4020
gagcatgagg aaaacagatt ggctcccaat gactgtggca gctatgggag ctcaacccct 4080
accacttttt attttcagtc tgaaagatac actcaaaagg agaagctggc cactgaatga 4140
gggggtgatg gcagttggac ttgtgagcat tctagctagt tctctcctta ggaatgatgt 4200
gcctatggct ggaccattag tggctggggg cttgctgata gcgtgctacg tcataactgg 4260
cacgtcagca gacctcactg tagaaaaagc agcagatgta acatgggagg aagaggccga 4320
gcaaacagga gtgtcccaca atttaatggt cacagttgat gatgatggaa caatgagaat 4380
aaaagatgac gagactgaga acatcttaac agtgctttta aaaacagcac tactaatagt 4440
atcaggcatc tttccatact ccatacccgc aacactgttg gtctggcata cttggcaaaa 4500
gcaaacccaa agatccggcg tcctatggga cgtacccagc cccccagaga cacagaaagc 4560
ggaactggaa gaaggggtct ataggatcaa acagcaagga atttttggga aaacccaagt 4620
gggggttgga gtacagaaag aaggagtttt ccacaccatg tggcatgtca caagaggggc 4680
agtgttgaca cacaatggga aaagactgga accaaactgg gctagcgtga aaaaagatct 4740
gatttcatac ggaggaggat ggagattgag tgcacaatgg aaaaaggggg aggaggtgca 4800
ggttattgcc gtagagcctg ggaagaaccc aaagaacttt caaaccatgc caggcatttt 4860
tcagacaaca acaggggaaa taggagcaat tgcactggat ttcaagcctg gaacttcagg 4920
atctcccatc ataaacagag agggaaaggt agtgggactg tatggcaatg gagtggttac 4980
aaagaatgga ggctatgtca gtggaatagc gcaaacaaat gcagaaccag atggaccgac 5040
accagagttg gaagaagaga tgttcaaaaa gcgaaatcta accataatgg atctccatcc 5100
tgggtcagga aagacgcgga aatatcttcc agctattgtt agagaggcaa tcaagagacg 5160
cttaaggact ctaattttgg caccaacaag ggtagttgca gctgagatgg aagaagcatt 5220
gaaagggctc ccaataaggt atcaaacaac tgcaacaaaa tctgaacaca caggaagaga 5280
gattgttgat ctaatgtgtc acgcaacgtt cacaatgcgc ttgctgtcac cagtcagggt 5340
tccaaactac aacttgataa taatggatga ggcccatttc acagacccag ccagtatagc 5400
ggctagaggg tacatatcaa ctcgtgtagg aatgggagag gcagccgcaa ttttcatgac 5460
agcaacaccc cctggaacag ctgatgcctt tcctcagagc aacgctccaa ttcaagatga 5520
agagagagac ataccggaac gctcatggaa ttcaggcaat gaatggatta ctgactttgt 5580
tgggaagaca gtgtggtttg tccctagcat caaagccgga aatgacatag caaactgctt 5640
gcggaaaaat ggaaaaaagg ttattcaact cagcaggaag acctttgaca cagaatatca 5700
aaagaccaaa ctgaatgatt gggactttgt ggtgacaaca gacatttcag aaatgggagc 5760
caatttcaaa gcagatagag tgatcgaccc aagaagatgt ctcaagccgg tgattttgac 5820
agatggaccc gagcgggtga tcctggctgg accaatgcca gtcaccgtag cgagcgctgc 5880
gcaaaggaga gggagagttg gcaggaaccc acaaaaagaa aatgaccagt acatattcat 5940
gggccagcct ctcaacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000
ggacaacatc aacacaccag aagggattat accagctctc tttgaaccag aaagggagaa 6060
gtcagccgcc atagacggcg aataccgcct gaagggtgag tccaggaaga ctttcgtgga 6120
actcatgagg aggggtgacc tcccagtttg gctagcccat aaagtagcat cagaagggat 6180
caaatataca gatagaaaat ggtgctttga tggagaacgt aataatcaaa ttttagagga 6240
gaatatggat gtggaaatct ggacaaagga aggagaaaag aaaaaactga gacctaggtg 6300
gcttgatgcc cgcacttatt cagatccttt agcactcaaa gaattcaagg attttgcagc 6360
tggcagaaag tcaatcgccc ttgatcttgt gacagaaata ggaagagtgc cttcacactt 6420
agcccacaga acgagaaacg ccctggataa tttggtgatg ctgcacacgt cagaacatgg 6480
cggtagggcc tacaggcatg cagtggagga actaccagaa acgatggaaa cactcttact 6540
cctgggactg atgatcttgt taacaggtgg agcaatgctc ttcttgatat caggtaaagg 6600
gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttatggat 6660
ggctgatgtc ccactccaat ggatcgcatc ggctatagtc ctggagtttt ttatgatggt 6720
gttgctcata ccagaaccag aaaagcagag aactccccaa gacaaccaac tcgcatatgt 6780
cgtgataggc atacttacat tggctgcaat agtagcggcc aatgaaatgg gactgttgga 6840
aactacaaag agagatttag gaatgtctaa agaaccaggt gttgtttctc caaccagcta 6900
tttggatgtg gacttgcacc cagcatcagc ctggacattg tacgccgtgg ccacaacagt 6960
aataacacca atgttgagac acaccataga gaattccaca gcaaatgtgt ctctggcagc 7020
catagctaac caggcagtgg tcctgatggg tttagacaaa ggatggccga tatcgaaaat 7080
ggacttgggc gtaccactat tggcactggg ttgctattca caagtgaacc cactaactct 7140
tgcagcggca gtacttttgc tagtcacaca ttatgcaatt ataggtccag gattgcaggc 7200
aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacggt 7260
ggatggaata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320
actaggacag gtcatgctcc tggttctgtg tgcagtccaa cttttattga tgagaacatc 7380
atgggccttg tgtgaagttc taaccctagc cacaggacca ataacaacac tctgggaagg 7440
atcacctggg aagttctgga acaccacgat agctgtttcc atggcgaaca tctttagagg 7500
gagctattta gcaggagctg ggcttgcttt ttctatcatg aaatcagttg gaacaggaaa 7560
gagaggaaca gggtcacaag gtgaaacctt aggagaaaag tggaaaaaga aattaaatca 7620
gttatcccgg aaagagtttg acctttacaa gaaatccgga atcaccgaag tggatagaac 7680
agaagccaaa gaagggttaa aaagaggaga aataacacac catgccgtgt ccagaggcag 7740
cgcaaaactt caatggttcg tggagagaaa catggtcatt cctgaaggaa gagtcataga 7800
cctaggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagttacaga 7860
agtgcgagga tacacaaaag gcggcccagg acacgaagaa ccagtaccta tgtctacata 7920
cggatggaac atagtcaagt taatgagtgg aaaggatgtt ttttatctgc cacctgaaaa 7980
gtgtgatacc ctattgtgtg acattggaga atcttcacca agcccaacag tggaagaaag 8040
cagaaccata agagttttga agatggttga accatggcta aagaacaacc agttttgcat 8100
taaagtattg aacccataca tgccaactgt gattgagcac ttagaaagac tacaaaggaa 8160
acatggagga atgcttgtga gaaatccact ctcacgaaac tccacgcacg aaatgtattg 8220
gatatccaat ggtacaggca atatcgtctc ttcagtcaac atggtatcca gattgctact 8280
gaacagattc acaatgacac acaggagacc caccatagag aaagatgtgg atctaggagc 8340
aggaacccga catgtcaatg cggaaccaga aacacccaac atggatgtca ttggggaaag 8400
aataaaaagg atcaaagagg agcatagttc aacatggcac tatgatgatg aaaatcctta 8460
caaaacgtgg gcttaccatg gatcctatga agtaaaagcc acaggctcag cctcctccat 8520
gataaatgga gtcgtgaaac tcctcacaaa accatgggat gtggtgccca tggtgacaca 8580
gatggcaatg acagatacaa ctccattcgg ccagcaaaga gtttttaaag agaaagtgga 8640
caccaggaca cctaggccca tgccaggaac aagaaaggtt atggagatca cagcggagtg 8700
gctttggagg accctgggaa ggaacaaaag acccagatta tgcacaaggg aggaattcac 8760
aaagaaggtc agaaccaacg cagctatggg cgctgtcttc acagaagaga accaatggga 8820
cagtgcgaga gctgctgttg aggacgaaga attttggaaa cttgtggaca gagaacgtga 8880
actccacaaa ctgggcaagt gtggaagctg cgtttacaac atgatgggca agagagagaa 8940
aaaacttgga gagtttggta aagcaaaagg cagtagggct atatggtaca tgtggttggg 9000
agccaggtac cttgagttcg aggcgctcgg attcctcaat gaagaccact ggttctcgcg 9060
tgaaaactct tacagtggag tagaaggaga aggactgcac aagctgggat acatcttgag 9120
agatatttcc aagatacccg gaggagccat gtatgctgat gacacagccg gttgggacac 9180
aagaataaca gaagatgacc tgcacaatga ggaaaaaatc acacagcaga tggaccctga 9240
acacaggcag ctagcgaacg ctatattcaa gctcacatac caaaacaaag tggtcaaagt 9300
ccaacgacca actccaaagg gcacggtaat ggacatcata tctaggaaag accaaagagg 9360
cagtggacag gtgggaactt atggtctgaa cacattcacc aacatggaag cccagctaat 9420
cagacaaatg gaaggagaag gcgtgttgtc aaaggcagac ctcgagaacc cccatccgct 9480
agagaagaaa attacacaat ggttggaaac taaaggagtg gaaaggttaa aaagaatggc 9540
catcagcggg gatgattgcg ttgtgaaacc aatcgacgac agattcgcca atgccctgct 9600
tgccctgaac gatatgggaa aggttagaaa ggacatacct caatggcagc catcaaaggg 9660
atggcatgat tggcaacagg tccccttctg ctcccaccac tttcatgaat tgatcatgaa 9720
agatggaaga aagttggtag ttccctgcag accccaggac gaactaatag gaagagcgag 9780
aatctcccaa ggagcaggat ggagccttag agaaactgca tgtctaggga aagcctacgc 9840
tcaaatgtgg gctctcatgt attttcacag aagagatctt agactagcat ccaacgccat 9900
atgttcagca gtaccagtcc actgggtccc cacgagcaga acgacatggt ctattcatgc 9960
tcaccatcag tggatgacta cagaagacat gcttactgtc tggaacaggg tgtggataga 10020
ggacaatcca tggatggaag acaaaactcc agtcacaacg tgggaagatg ttccatatct 10080
agggaagaga gaagaccaat ggtgcggatc actcataggt ctcacttcca gagcaacctg 10140
ggcccagaac atactcacag caatccaaca ggtgagaagc ctcataggca atgaagagtt 10200
tctggactac atgccttcga tgaagagatt caggaaggag gaggagtcag agggagccat 10260
ttggtaaaag caggaggtaa actgtcaggc cacattaagc cacagtacgg aagaagctgt 10320
gcagcctgtg agccccgtcc aaggacgtta aaagaagaag tcaggcccaa aagccacggt 10380
ttgagcaaac cgtgctgcct gtagctccgt cgtggggacg taaagcctgg gaggctgcaa 10440
accgtggaag ctgtacgcac ggtgtagcag actagtggtt agaggagacc cctcccatga 10500
cacaacgcag cagcggggcc cgagcactga gggaagctgt acctccttgc aaaggactag 10560
aggttagagg agaccccccg caaacaaaaa cagcatattg acgctgggag agaccagaga 10620
tcctgctgtc tcctcagcat cattccaggc acagaacgcc agaaaatgga atggtgctgt 10680
tgaatcaaca ggttctagt 10699
<210> 42
<211> 10648
<212> DNA
<213> Dengue virus
<220>
<221> misc_feature
<222> (1)..(10648)
<223> LAV4
<400> 42
agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60
ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120
tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180
ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240
tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaaa 300
gatggggaca gttgaagaaa aataaggcca tcaggatact gattggattc aggaaggaga 360
taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct 420
tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480
tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540
gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600
ccttactggt caataccgaa cctgaagaca ttgattgctg gtgcaatctc acgtctacct 660
gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720
ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780
aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggattcg 840
cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900
tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgta ggagtaggaa 960
acagagactt tgtggaagga gtctcaggtg gagcatgggt cgatctggtg ctagaacatg 1020
gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1080
caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1140
taaccacggc aacaagatgt ccaacgcaag gagagcctta tctaaaagag gaacaagacc 1200
aacagtacat ctgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1260
ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1320
atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1380
cccatgcagt aggaaatgac acatccaatc atggagttac agccacgata actcccaggt 1440
caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1500
ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggcttg 1560
tgcataagca atggtttttg gatctacctc taccatggac agcaggagca gacacatcag 1620
aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1680
aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1740
cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatctcaag tgcaaagtcc 1800
gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttctcaa 1860
ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1920
gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtgaacaag aaaaaagtgg 1980
ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgca accaacatag 2040
agttagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agtgcattaa 2100
cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2160
gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2220
tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2280
ttggaggagt ctcatggatg attagaatcc taattgggtt cctagtgttg tggattggca 2340
cgaactcaag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2400
ttctgggctt cacagttcaa gcagacatgg gttgtgtggt gtcatggagt gggaaagaat 2460
tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520
aatttcaacc ggagtcccca gcgagactag cgtctgcaat attgaatgcc cacaaagatg 2580
gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2640
acgagctaaa ttatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2700
tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgaat gatctgaaat 2760
attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2820
ttttaataga cggaccagac acctccgaat gccccaatga acgaagagca tggaactttc 2880
ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2940
aaggaagttc agaagtgtgt gaccacaggt taatgtcagc ggcaattaaa gatcagaaag 3000
ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3060
agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acattgtgga 3120
gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3180
accacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3240
tagagataga ctttggagaa tgccccggaa caacagtcgc aattcaggag gattgtgacc 3300
atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3360
gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3420
tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtaacggccg 3480
gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3540
aatgcttgag gagaagagtc actaggaaac acatgatatt ggttgtggtg atcactcttt 3600
gtgccatcat cctaggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3660
gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3720
agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780
cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3840
aactcattga tggaatatca ctggggctaa ttttgctaaa aatagtgaca cattttgaca 3900
acacccaagt gggaacctta gccctttcct tgaccttcat aagatcaaca atgccattgg 3960
tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020
ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080
cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140
ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200
agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260
tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaatgtg cagtgggatg 4320
aaatggcaga cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct 4380
ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4440
tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4500
tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4560
ctcaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttatttggga 4620
aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4680
caagaggatc agtgatctgc catgagactg ggagattgga gccatcttgg gctgacgtca 4740
ggaatgacat gatatcatac ggtgggggat ggagacttgg agacaaatgg gacaaagaag 4800
aagatgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4860
ccggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4920
gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4980
gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5040
agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5100
tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5160
aaaggaggct gcgaaccttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220
aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5280
gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa 5340
ccagagttcc aaattacaac ctcatagtga tggatgaagc acatttcacc gatccttcta 5400
gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5460
tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5520
aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5580
actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5640
attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700
agtatccaaa aacgaaactc acggactggg attttgtggt cactacagac atatctgaaa 5760
tgggggccaa ttttagagct gggagagtga tagaccctag gagatgcctc aagccagtta 5820
tcctaacaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5880
gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940
ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6000
tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060
gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6120
ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6180
ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaaggaat aaccaaattt 6240
tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6300
caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat tttaaggagt 6360
ttgctagtgg aaggaagagc ataactctcg acatcctaac agagattgcc agtttgccaa 6420
cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6480
aaagaggagg gagggcctac caacacgccc tgaacgaact cccggagtca ctggaaacac 6540
ttatgcttgt agctttacta ggtgctatga cagcaggtat cttcctgttt ttcatgcaag 6600
ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgt 6660
tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6720
tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780
tctacgtcat attgaccatt ctcaccatta ttggtctcat agcagccaac gagatggggc 6840
tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900
tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacawttc 6960
tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7020
ttgccaacca ggcggccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7080
acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaactttga 7140
cagcatcctt agtcatgctt tcagtccatt atgcaataat aggtccagga ttgcaggcaa 7200
aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaac cccacggtgg 7260
acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320
tagggcaggt catgctactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380
gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7440
acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7500
gttacctggc gggagctgga ctggcttttt cactcataaa gaatgyacaa acccctagga 7560
ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620
takacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680
aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740
gtaagattag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7800
ttggctgtgg gagaggagga tggtcttatt acatggcgac gctcaagaac gtgactgaag 7860
tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7920
gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7980
tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8040
gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8100
tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8160
aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8220
gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8280
tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8340
caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatt attgggagaa 8400
ggcttcagcg attgcaagag gagcacaaag aaacctggca ttatgatcag gaaaacccat 8460
acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8520
tggtgaacgg ggtagtaaaa ctgctaacaa aaccttggga tgtggttcca atggtgaccc 8580
agttagccat gacagacaca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8640
ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8700
ggctgtgggc cctccttggg aagaagaaaa atcccagact gtgcacaagg gaagagttca 8760
tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8820
catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880
ccctacacca ggaagggaaa tgtgaatcgt gtgtctacaa catgatggga aaacgtgaga 8940
aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000
gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9060
gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9120
aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9180
caagaatcac tgaggatgac cttcaaaatg aagaactgat cacggaacag atggcccccc 9240
accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9300
tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaaa gaccaaagag 9360
gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9420
tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9480
tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9540
caatcagtgg agacgattgc gtggtgaagc ccctggatga gaggtttggc acttccctcc 9600
tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9660
gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atcttcatga 9720
aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780
gaatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840
cccagatgtg gtcgctcatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9900
tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9960
ctcatcatca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag 10020
aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc 10080
tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct 10140
gggcgaagaa cattcacacg gccataaccc aggtcagaaa cctgatcgga aaagaggaat 10200
acgtggatta catgccagta atgaaaagat acagcgctcc ttcagagagt gaaggagttc 10260
tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggctt 10320
gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtgaaatc cctagggagg 10380
ccatgcgcca cggaagctgt acgcgtggca tattggacta gcggttagag gagacccctc 10440
ccatcactga caaaacgcag caaaaggggg cccgaagcca ggaggaagct gtactcctgg 10500
tggaaggact agaggttaga ggagaccccc ccaacacaaa aacagcatat tgacgctggg 10560
aaagaccaga gatcctgctg tctctgcaac atcaatccag gcacagagcg aagcaagatg 10620
gattggtgtt gttgatccaa caggttct 10648
Claims (17)
- 서열번호 38에서 서열번호 38의 데옥시티미딘 대신 우리딘을 포함하는 RNA 서열을 포함하는 약독화된 뎅기-2 바이러스 생균주(live attenuated dengue-2 virus strain)로서, 서열번호 38을 기준으로 상기 RNA 서열의 적어도 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치의 뉴클레오티드가 돌연변이되고, 57, 524, 2055, 2579, 4018, 5547, 6599 및 8571번 위치의 뉴클레오티드는 돌연변이되지 않았으며,
상기 RNA 서열의 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치에서 일어나는 돌연변이된 뉴클레오티드는 각각 C, A, A, C, A, A 및 G인, 약독화된 뎅기-2 바이러스 생균주. - 제 1항에 있어서, 상기 RNA 서열은 서열번호 38의 서열을 기준으로, 736, 1619, 1638, 2520, 4723, 5062, 9191, 9222, 10063 및 10507번 위치에서 돌연변이를 포함하며, 736, 1619, 1638 , 2520, 4723, 5062, 9191, 9222, 10063 및 10507번 위치에서 일어난 변이된 뉴클레오티드가 각각 C, A, G, A, A, C, A, G, A 및 G 인, 약독화된 뎅기-2 바이러스 생균주.
- 제 1항에 있어서, 상기 RNA 서열은 서열번호 38에 의하여 코딩되는 아미노산을 기준으로, 주어진 위치에서 코딩하고 있는 아미노산을 변이시키지 않고 해당 코돈 위치에 하나 이상의 뉴클레오티드 치환을 더 포함하는 것인, 약독화된 뎅기-2 바이러스 생균주.
- 제 1항에 있어서, 상기 RNA 서열은 서열번호 1에서 서열번호 1의 데옥시티미딘 대신 우리딘을 포함하는 RNA 서열과 5 돌연변이 이하의 차이를 가지는 것인, 약독화된 뎅기-2 바이러스 생균주.
- 제 1항에 있어서, 서열번호 1에서 서열번호 1의 데옥시티미딘 대신 우리딘을 포함하는 RNA 서열을 포함하는 약독화된 뎅기-2 바이러스 생균주.
- 제 1항 내지 제 5항 중 어느 한 항에 따른 약독화된 뎅기-2 바이러스 생균주를 약제학적으로 허용가능한 담체 내에 포함하는 면역원성 조성물.
- 제 6항에 있어서, 1가(monovalent) 백신 조성물인, 면역원성 조성물.
- 제 6항에 있어서, 다가(multivalent) 뎅기 백신 조성물인, 면역원성 조성물.
- 제 8항에 있어서, 서열번호 39에서 서열번호 39의 데옥시티미딘 대신 우리딘을 포함하는 RNA를 포함하는 약독화된 뎅기-1 바이러스 생균주를 포함하는 면역원성 조성물.
- 제 6항에 있어서, 상기 약독화된 뎅기-2 바이러스 생균주를 10 내지 105 CCID50로 포함하는, 면역원성 조성물.
- 서열번호 1의 DNA 서열 또는 서열번호 1에서 서열번호 1의 데옥시티미딘 대신 우리딘을 포함하는 RNA 서열을 포함하는 핵산.
- 서열번호 1에 의해 코딩되는 폴리단백질.
- 서열번호 38에서, 서열번호 38을 기준으로 뉴클레오티드 서열의 적어도 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치의 뉴클레오티드가 돌연변이되고, 상기 뉴클레오티드 서열의 57, 524, 2055, 2579, 4018, 5547, 6599 및 8571번 위치의 뉴클레오티드는 돌연변이되지 않은 서열을 가지는 뉴클레오티드 서열로부터 만들어지는 RNA를 포함하는 뎅기-2 바이러스 균주로서,
상기 뉴클레오티드 서열의 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치에서 일어나는 돌연변이된 뉴클레오티드는 각각 C, A, A, C, A, A 및 G인, 뎅기-2 바이러스 균주. - 서열번호 38에서, 서열번호 38의 데옥시티미딘 대신 우리딘을 포함하는 RNA서열을 가지는 RNA 분자로서,
서열번호 38을 기준으로 상기 RNA 서열의 적어도 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치의 뉴클레오티드가 돌연변이되고, 57, 524, 2055, 2579, 4018, 5547, 6599 및 8571번 위치의 뉴클레오티드는 돌연변이되지 않았으며,
상기 RNA 서열의 736, 1619, 4723, 5062, 9191, 10063 및 10507번 위치에서 일어나는 돌연변이된 뉴클레오티드는 각각 C, A, A, C, A, A 및 G인, RNA 분자. - 서열번호 1에서, 서열번호 1의 데옥시티미딘 대신 우리딘을 포함하는 RNA 서열을 가지는 RNA 분자.
- 삭제
- 삭제
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| FR2909286B1 (fr) * | 2006-12-01 | 2012-06-08 | Sanofi Pasteur | Methode d'immunisation contre les 4 serotypes de la dengue |
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| CN103382509A (zh) * | 2013-07-17 | 2013-11-06 | 浙江国际旅行卫生保健中心 | 逆转录-环介导等温扩增检测登革2型病毒的试剂及检测方法 |
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| US7641908B2 (en) | 2010-01-05 |
| US8067566B2 (en) | 2011-11-29 |
| CA2611954C (en) | 2014-01-28 |
| JP5075120B2 (ja) | 2012-11-14 |
| MY144542A (en) | 2011-09-30 |
| KR20140019034A (ko) | 2014-02-13 |
| JP2008546383A (ja) | 2008-12-25 |
| EP1891210A1 (en) | 2008-02-27 |
| BRPI0613287A2 (pt) | 2010-12-28 |
| TWI375722B (en) | 2012-11-01 |
| US20150031857A1 (en) | 2015-01-29 |
| DE602006015807D1 (de) | 2010-09-09 |
| WO2006134443A1 (en) | 2006-12-21 |
| TW200738881A (en) | 2007-10-16 |
| US8795688B2 (en) | 2014-08-05 |
| KR20080018271A (ko) | 2008-02-27 |
| US20070026016A1 (en) | 2007-02-01 |
| CN101238209A (zh) | 2008-08-06 |
| EP1891210B1 (en) | 2010-07-28 |
| CA2611954A1 (en) | 2006-12-21 |
| US20120083585A1 (en) | 2012-04-05 |
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