猪冠状病毒疫苗

文档序号:1785315 发布日期:2019-12-06 浏览:16次 >En<

阅读说明:本技术 猪冠状病毒疫苗 (Porcine coronavirus vaccine ) 是由 S·阿克曼 J·R·赫尔曼 L·A·赫尔南德斯 L·A·霍布斯 A·V·耶尔 S·奥康纳 于 2018-01-26 设计创作,主要内容包括:本发明涉及用于保护猪不受与冠状病毒感染相关的疾病的疫苗,所述冠状病毒感染包括猪流行性腹泻病毒(PEDV)和/或猪丁型冠状病毒(PDCoV)。疫苗通常包括灭活/杀死的PEDV(例如,化学灭活的PED病毒)、和/或重组PEDV抗原、和/或佐剂灭活/杀死的PDCoV(例如,化学灭活的PDCoV病毒)、和/或重组PDCoV抗原和佐剂。还提供了用于保护猪不受与PEDV和/或PDCoV相关的疾病的方法,以及产生猪流行性腹泻病毒和/或猪丁型冠状病毒疫苗的方法。(The present invention relates to vaccines for protecting pigs against diseases associated with coronavirus infection, including Porcine Epidemic Diarrhea Virus (PEDV) and/or porcine delta coronavirus (PDCoV). The vaccine typically comprises inactivated/killed PEDV (e.g., chemically inactivated PED virus), and/or recombinant PEDV antigen, and/or adjuvant inactivated/killed PDCoV (e.g., chemically inactivated PDCoV virus), and/or recombinant PDCoV antigen and adjuvant. Also provided are methods for protecting pigs from diseases associated with PEDV and/or PDCoV, and methods of producing porcine epidemic diarrhea virus and/or porcine delta coronavirus vaccines.)

猪冠状病毒疫苗

序列表

本申请包含根据37 C.F.R.1.821–1.825的序列表。大小为480kb、名称为10-0176-WO-SEQ的序列表的ASCII文本文件的内容于2017年1月23日创建,并且经由EFS-Web以电子方式随同提交,该申请整体引入本文作为参考。

技术领域

本发明涉及免疫原性组合物,其保护猪免于由特异性的猪流行性腹泻病毒(PEDV)疫苗和猪丁型冠状病毒(PDCoV)引起的疾病,以及提供PEDV和PDCoV抗原两者的组合疫苗。由于小于10日龄仔猪中的高死亡率(高达100%),经常一起存在的流行性肠道疾病是美国养猪业的主要经济关注点。

背景技术

猪流行性腹泻病毒是有包膜的正义单链RNA病毒,其引起猪中的急性腹泻、呕吐和脱水。它首先在欧洲得到鉴定,但在许多亚洲国家,包括朝鲜、中国、日本、菲律宾和泰国,已变得越来越成问题。在2013年4月,PEDV在美国中西部的猪中出现,迅速蔓延到全国各地。到2013年10月,PEDV在18个州的猪群中检测到。PEDV感染的经济影响已经很大。已鉴定了PEDV的北美分离株(Huang等人2013;Stevenson等人2013),然而,在美国没有商购可得的完全批准的疫苗。相应地,一直需要开发能够保护猪不受与PEDV相关的疾病的疫苗。开发针对新出现的北美PEDV毒株有效的疫苗将是有利的,所述疫苗可以经由粘膜途径(经口或鼻内)以及经由肠胃外方法(例如,肌内、皮下或静脉内)施用。

PEDV是甲型冠状病毒属(Alphacoronavirus)的冠状病毒亚科(Coronavirinae)的成员(Bridgen等人1993),并且在1971年首次在英国得到鉴定,并且后来在其它国家如比利时、中国、匈牙利、意大利、日本、朝鲜和泰国发现(Oldham J.1972;Pensaert和De BouckP.1978;Chen等人2008;Nagy等人1996;Martelli等人2008;Takahashi等人1983;Chae等人2000;和Puranaveja等人2009)。该家族的其它成员包括猪呼吸道冠状病毒(PRCV)、血凝性脑脊髓炎冠状病毒(PHE)和传染性胃肠炎病毒(TGEV)。尽管PEDV和TGEV病毒是相关的并且临床体征非常相似,但不存在免疫交叉保护。

PEDV是具有大约28kb正义单链RNA基因组的有包膜病毒,具有5'帽和3'多腺苷酸化尾。(Pensaert和De Bouck P.1978)。基因组包含5'非翻译区(UTR)、3'UTR和至少7个开放读码框(ORF),其编码四种结构蛋白(刺突(S)、包膜(E)、膜(M)和核衣壳(N))和三种非结构蛋白(复制酶1a和1b以及ORF3);这些以5’–复制酶(1a/1b)-S-ORF3-E-M-N-3’的次序排列在基因组上(Oldham J.1972;和Bridgen等人1993)。表征的前三种新出现的北美PEDV基因组序列:Minnesota MN(GenBank:KF468752.1)、Iowa IA1(GenBank:KF468753.1)和Iowa IA2(GenBank:KF468754.1),具有28,038个核苷酸(nt)的相同大小,排除聚腺苷尾,并且与原型PEDV CV777毒株(GenBank:AF353511.1)共享基因组组构。这三种北美PEDV序列共享99.8%至99.9%的核苷酸同一性。特别地,毒株MN和IA2跨越整个基因组仅具有11个核苷酸差异。

PEDV S蛋白是由1,383个氨基酸(aa)组成的I型糖蛋白。基于其与其它冠状病毒的S蛋白的同源性,S蛋白可以分为S1(1-789aa)和S2(790-1,383aa)结构域(Chang等人;2002;Cruz等人,1994;Godet等人1994;Jackwood等人2001;Sturman和Holmes;1984;以及Sun等人2008)。冠状病毒中的S蛋白是表面抗原,其在调节与宿主细胞受体糖蛋白的相互作用以介导病毒进入,并且刺激天然宿主中的中和抗体的诱导中起作用。因此,S糖蛋白是用于开发针对PEDV的有效疫苗的主要靶。

PEDV M蛋白是最丰富的包膜组分,其在病毒组装过程中起重要作用,并且还诱导中和病毒的抗体。同样地,对于核衣壳提供结构基础、与病毒粒子RNA结合PEDV N蛋白,对于诱导细胞介导的免疫也可能是重要的(Saif,L.1993)。

PEDV中唯一的辅助基因是ORF3。虽然辅助基因一般在野毒株中得到维持,但ORF3的改变被认为影响毒力;细胞培养适应已用于改变ORF3基因,以便减少毒力(Song等人2003)。事实上,通过对ORF3基因的调查,研究人员已描绘了自2006年以来中国免疫猪群中的PEDV新基因群的出现。这些毒株的系统发育研究和PEDV在中国的地理重现已证实引起破坏性肠道疾病的那些野毒株在ORF3中与欧洲毒株和疫苗株在遗传上不同(Park等人2011)。

众所周知确实存在具有不同水平毒力的PEDV的不同毒株。在20世纪80年代和90年代期间,PEDV在整个欧洲在国家如比利时、英国、德国、法国、荷兰和瑞士流行。欧洲报告病例的频率随后逐渐减少和/或由PEDV引起的疾病对于开始疫苗的商业开发不具有足够的经济重要性(Song和Park 2012)。自20世纪80年代以来,中国已记录了PEDV的爆发,与大规模腹泻爆发相关的自2010年以来出现的PEDV变异株已变得更急性和严重。因此,疫苗开发试验主要在亚洲国家完成(Song和Park 2012)。自2010年以来出现的变异据报道在哺乳仔猪中具有80-90%的发病率和50-90%的死亡率(Bi等人2012;Pan等人2012;和Li等人1012)。最近的证据提示,中国新出现的PEDV毒力形式可能是活疫苗株进化的结果(Chen等人2010)。

作为影响猪肠的肠道疾病,PEDV经由粪-口暴露传播。受污染的卡车和设备是幼稚动物的常见感染源。PEDV感染的临床体征类似于传染性胃肠炎病毒(TGEV)感染(Pijpers等人1993)。在3周龄和更年幼的猪中,早在PEDV感染后24小时可以看到临床体征(包括急性水样、腹泻、呕吐和脱水),导致100%的死亡率。受PEDV感染的饲养者和生长猪以及母猪和公猪可能发展腹泻和呕吐。动物也可以显示厌食症的体征,并且可能是嗜睡的。对老龄猪的全面影响尚未确定,但很可能饲料效率减少、上市天数增加、以及受感染动物对继发感染的易感性。对于母猪,身体状况的减少可能负面影响繁殖性能。报告已指示存在PEDV可以变成北美兽群中的地方性流行病的迹象,导致持续性腹泻和其它挑战。

感染PEDV的动物肠道中的总体和组织学变化在美国与在中国观察到的那些是相似的;基本上该病毒破坏猪肠的绒毛,使得无法吸收营养素。Huang等人2012报道死于明尼苏达州和爱荷华州爆发的疾病的动物具有局限于小肠的肉眼可见的病理性病变,并且小肠的特征在于由黄色流体扩张的半透明薄壁。组织学评估揭示具有绒毛钝化和融合的小肠区域以及固有层绒毛的最低限度成淋巴细胞浸润。

Huang等人2013表征了来自美国的输出爆发的三种不同的PEDV毒株-一种来自明尼苏达州,并且两种来自爱荷华州,分别命名为MN(GenBank登录号:KF468752)以及IA1(GenBank登录号:KF468753)和IA2(GenBank登录号:KF48754)。Huang的系统发育调查将PEDV毒株分组为两个不同的基因群,分别命名为基因群1(G1)和基因群2(G2)。刺突基因的N末端结构域(NTD)中的显著变化区分了基因群1和2。Huang等人2013提出N末端结构域(NTD)中的第二缺失区域(DR2)看来具有比DR1更高的抗原变化程度,提示新出现的北美毒株可能与G1a疫苗株“在抗原上”更不相关。

基因群1包括至少三个簇1a、1b和R。亚群1a包括早期欧洲、中国和朝鲜分离株,例如原型CV777毒株(比利时,1978,GenBank:AF353511.1)和毒株LZC(甘肃,中国,2006;GenBank:EF185992)和SM98(朝鲜,1998;GenBank:GU937797.1)。亚组1b含有5个毒株—一个来自韩国(DR13减毒疫苗株,GenBank:JQ023162.1),并且其它来自中国,通过nsp3中常见的“遗传标记”8-aa缺失和在C末端处的大ORF3缺失联系。组“R”与其它基因群的重组体相关。然而,新近出现的PEDV毒株,包括自2010年以来在中国和自2013年以来在北美出现的那些,属于基因群G2a。中国毒株AH2012(GenBank登录号:KC210145)和北美毒株共享几个独特的核苷酸变化,并且在基因群2a中聚簇在一起。对AH2012的核苷酸同一性对于毒株MN和IA2为99.6%,并且对于毒株IA1为99.5%。研究人员已推测,AH2012样病毒可能传播到中国东部地区,然后运输至美国,并且很可能是最接近北美株的祖先。基因群2a的成员与基因群1a的原型PEDV毒株CV777仅共享大约96.9%的相似性(Bridgen等人1993;Huang等人2013;GenBank:AF353511.1)。像这样,基于历史CV777衍生的G1a毒株或DR13衍生的G1b毒株的减毒PEDV疫苗可以与新近出现的中国和北美G2a PEDV毒株在抗原上较不相关,并且因此可能是弱疫苗候选物。

密切相关的北美分离株US/Colorado/2013(GenBank登录号:KF272920.1)也已通过Marthaler等人,2013得到报道。如同上述北美分离株,CO/13的完全PEDV基因组与GenBank中可获得的其它完全PEDV基因组具有96.5%至99.5%的核苷酸同一性,与中国毒株AH2012(GenBank登录号KC210145)具有最高的核苷酸同一性(99.5%)。它是2a基因群的成员。通过CO/13的完全基因组与PEDV参考毒株CV777的完全基因组的比较,证实CO/13含有1-nt***(在位置48处)和5'UTR中5个核苷酸的缺失(在位置73和83至86处)。这种北美病毒显示出在基因组位置20,696和21,125处的S1内增加的差异,与几种***/缺失仅共享82%的核苷酸同一性。

已开发了在其基因组序列、递送模式和功效方面不同的几种PEDV疫苗。欧洲CV777毒株的细胞培养适应已在其中PEDV爆发严重的亚洲国家使用。这些自20世纪90年代以来一直在使用。

在20世纪80年代早期,日本研究人员从受感染猪的腹泻中分离出致病的PED病毒株83P-5。Kusanagi等人1989在Vero细胞中分离且适应了该毒株。自1997年以来,适应PEDV(P-5V)(83P-5)的细胞培养的减毒病毒疫苗已在日本的母猪中使用。第100次传代的83P-5毒株在日本被Nisseiken Co.,Ltd.(Sato等人2011)批准用作减毒PEDV疫苗。已报道83P-5毒株的适应和减弱显示S蛋白的细胞外部分中的突变,与减毒DR13毒株的那种具有序列相似性(Sato等人2011;参见在第100次传代时的毒株83P-5刺突基因序列,GenBank:AB548621.1)。尽管这种后来的日本疫苗被视为有效的,但并非所有母猪都能够对其仔猪传递免疫力(Usami等人1998)。日本毒株和欧洲毒株是基因群G1a或G1b的成员。如上文讨论的,与新近出现的中国品系的基因群2a相比,这些减毒的疫苗株与趋异的北美毒株更不相关。

用减毒的朝鲜PEDV毒株DR13(传代水平100)(GenBank:JQ023162.1)(基因群G1b的成员)的经口疫苗接种已显示作为疫苗是有效的。自2004年以来,该病毒株在韩国被批准并且用作经口疫苗,并且在2011年在菲律宾登记且商业化(Song和Park2012)。然而,据报道减毒的DR13不显著改变受攻击仔猪中病毒脱落的持续时间-这提示免疫保护是不完全的。此外,用高度减毒的PEDV经口免疫仅在非常高剂量的疫苗中赋予保护(Song和Park 2012)。

其它已知的疫苗包括 PT-100(ChoongAng Vaccine Laboratories,韩国),组合杀死的PEDV和TGEV疫苗,以及 PED,杀死的PEDV疫苗。通过ChoonAngVaccine Laboratories提供的毒株和亚型是未知的。另外,另一家韩国公司KomipharmInternational Co.提供以商品名销售的一系列杀死的、活的和组合疫苗,其包括基因群G1a的PEDV毒株SM98P。中国的Qilu Animal Health Products Factory也在中国上市了含有PEDV和TGEV的组合杀死的疫苗,其毒株和亚型未知。

新近出现的猪丁型冠状病毒(porcine deltacorona virus)(PDCoV),也称为猪丁型冠状病毒(swine deltacorona virus)SDCoV或SDCV,并且在本文中可互换使用,类似于猪流行性腹泻病毒(PEDV),并且经常牵涉新生动物中的发病率,且促成该疾病对养猪业的全球传播和影响。PDCoV在PEDV之后的短时间内影响美国,具有类似的发病模式。在美国,与PEDV不同,丁型冠状病毒看来不引起仔猪中的死亡率,但引起仔猪中的急性发病率包括严重腹泻、呕吐和重量减轻。虽然比PEDV感染更温和,但PDCoV感染可以导致新生儿中30-40%的死亡率。在具有PEDV、轮状病毒和TGEV的共感染中可以看到更严重的疾病(Marthaler等人,2014a;和Wang等人2014)。另外,在2015年5月,在美国检测到第三种新试剂,猪正呼肠孤病毒(PORV),当在共感染中存在时潜在地促成疾病严重性。虽然需要PDCoV和其它共感染试剂的发病机制和临床牵涉的更多了解,但PEDV和PDCoV的这种最近共出现已潜在地增强了疾病对畜群的严重性和影响。

猪丁型冠状病毒(PDCoV)是冠状病毒科(Coronaviridae)、丁型冠状病毒属(deltacoronavirus)的成员。该病毒在2009年收集的猪直肠拭子样品中首次在香港SAR得到鉴定(Woo,P.C.等人,Discovery of seven novel Mammalian and aviancoronaviruses in the genus deltacoronavirus supports bat coronaviruses as thegene source of alphacoronavirus and betacoronavirus and avian coronavirusesas the gene source of gammacoronavirus and deltacoronavirus.J Virol,2012.86(7):第3995-4008页),但不知道与任何疾病有关。SDCV,在本文中称为PDCoV,在2014年2月在俄亥俄州和印第安纳州首次在美国猪群中检测到,并且迅速传播到其它州(Hu,H.等人,Isolation and Characterization of Porcine Deltacoronavirus from Pigs withDiarrhea in the United States.J Clin Microbiol,2015.53(5):第1537-1548页;Chen,Q.等人,Pathogenicity and pathogenesis of a United States porcinedeltacoronavirus cell culture isolate in 5-day-old neonatal piglets.Virology,2015.482:第51-59页;Li,G.等人,Full-Length Genome Sequence of PorcineDeltacoronavirus Strain USA/IA/2014/8734.Genome Announc,2014.2(2);Marthaler,D.等人,Rapid detection,complete genome sequencing,and phylogenetic analysisof porcine deltacoronavirus.Emerg Infect Dis,2014a.20(8):第1347-50页;Marthaler,D.等人,Complete Genome Sequence of Strain SDCV/USA/Illinois121/2014,a Porcine Deltacoronavirus from the United States.Genome Announc,2014b.2(2);Wang,L.,B.Byrum和Y.Zhang,Detection and genetic characterization ofdeltacoronavirus in pigs,Ohio,USA,2014.Emerg Infect Dis,2014.20(7):第1227-30页;Jung,K.等人,Pathogenicity of 2porcine deltacoronavirus strains ingnotobiotic pigs.Emerg Infect Dis,2015.21(4):第650-4页)。PDCoV也在2014年4月在朝鲜(Lee,S.和C.Lee,Complete Genome Characterization of Korean PorcineDeltacoronavirus Strain KOR/KNU14-04/2014.Genome Announc,2014.2(6))、中国大陆(Dong,N.等人,Porcine Deltacoronavirus in Mainland China.Emerg Infect Dis,2015.21(12):第2254-5页;和Song,D.等人,Newly Emerged Porcine DeltacoronavirusAssociated With Diarrhoea in Swine in China:Identification,Prevalence andFull-Length Genome Sequence Analysis.Transbound Emerg Dis,2015.62(6):第575-80页)、以及日本(ISERPD 2015)检测到。

在鉴定后,三个研究小组已确认,用来自临床病例的细胞培养物衍生材料和/或组织匀浆物的感染导致已知菌和/或常规仔猪中的急性胃肠道临床体征(Chen等人,2015;Ma,Y.等人,Origin,evolution,and virulence of porcine deltacoronaviruses in theUnited States.MBio,2015.6(2);和Vitosh-Sillman,S.等人Histopathological andimmunohistochemical characterization of pigs experimentally infected withporcine deltacoronavirus.in American Association of SwineVeterinarians.2015.Orlando,FL)。PDCoV已显示在临床体征发作后8-9天引起分娩室中的母猪和仔猪中的急性腹泻(Li等人2014),连同呕吐和脱水(Ma等人2015)。该病毒具有与猪肠道冠状病毒(PEDV)、传染性胃肠炎冠状病毒(TGEV)和猪正呼肠孤病毒(PORV)相似的临床体征和症状。

通过Marx等人(WO201600757A2)的最近公开内容描述了据称保护猪免于由猪流行性腹泻病毒(PEDV)和PDCoV引起的疾病的免疫原性组合物。然而,很明显,当用减毒的欧洲INDEL毒株Calaf14攻击(在对照动物中仅引起23.8%的死亡率)时,基于北美科罗拉多株病毒的Marx等人的PEDV疫苗仅是部分保护性的(例如,当比较死亡率、重量减轻或腹泻的临床症状中的适度减少时)。此外,虽然Marx等人还描述了丁型冠状病毒疫苗,用单价PDCoV疫苗或二价PEDV和PDCoV疫苗未证实疾病的临床症状的保护或减少

因此,需要PEDV和PDCoV疫苗,其能够减少由PEDV和PDCoV引起的疾病的临床体征,并且在免疫动物中诱导保护性免疫,包括减少免疫动物中的病毒脱落。

发明内容

本发明提供了克服本领域缺陷的免疫原性组合物、疫苗和相关方法。本发明涉及免疫原性组合物,其包括冠状病毒科的有包膜(+)单链RNA病毒的灭活/杀死和/或重组形式:猪流行性腹泻病毒、或PEDV和/或猪丁型冠状病毒、或PDCoV。特别地,该申请提供了免疫原性组合物,其减少了猪中与PEDV和PDCoV感染相关的疾病的临床症状。本文PDCoV分离株(NSVL)(SEQ ID NO:1和2)以及分离株PDCoV 2.0307和PDCoV 5.0327(分别为SEQ ID NO:5、6、9和10)具有与其它北美和亚洲毒株的那些相似的遗传谱。本文PEDV分离株BI1251-125-10(在本文中称为“125-10”)(SEQ ID NO:29、32)是强毒的北美RNA病毒株,具有与基因群2a报道的其它北美PEDV的那些相似的遗传谱。PEDV衍生物分离株“125-10p30”(SEQ ID NO:34、36)是传代减毒病毒,也具有与北美基因群2a一致的遗传谱。

本发明的免疫原性组合物和疫苗包含灭活/杀死的PDCoV(例如,化学灭活的NSVL分离株(SEQ ID NO:1和2),和/或分离株PDCoV 2.0307和PDCoV 5.0.327(分别为SEQ IDNO:5和6以及SEQ ID NO:9和10),和/或灭活/杀死的PEDV(例如,化学灭活的PEDV分离株“125-10”或“125-10p30”(分别为SEQ ID NO:29或32和SEQ ID NO:33或36)),并且通常还包括佐剂。疫苗还可以包括其它组分,例如防腐剂、抗微生物剂、稳定剂例如可以增加疫苗的贮存期限的稳定剂、乳状液以及针对其它猪病原体的抗原。

本发明的免疫原性组合物和疫苗包含刺突抗原,其经由表达PDCoV和/或PEDV刺突蛋白,例如PDCoV刺突蛋白(例如,包含编码氨基酸序列SEQ ID NO:4、8、12、18或28的核酸序列SEQ ID NO:3、7、11、17或27)、和/或PEDV刺突核酸序列(包含编码氨基酸序列SEQ ID NO:31、35、47或53的SEQ ID NO:30、34、46或52)的重组杆状病毒在昆虫细胞的一个非限制性实例中表达,并且通常还包括佐剂。疫苗还可以包括其它组分,例如防腐剂、稳定剂以及针对其它猪病原体的抗原。

适用于本发明的优选PDCoV和/或PEDV刺突核酸序列是编码刺突多肽的多核苷酸,所述多核苷酸与PDCoV刺突多肽SEQ ID NO:3、7、11、17或其功能片段,和/或PEDV刺突SEQID NO:30、34、46或52或其功能片段至少具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%或89%,更优选至少90%,91%,92%,93%或94%,且最优选至少95%、96%、96.1%、96.2%、96.3%、96.4%、96.5%、96.6%、96.7%、96.8%、96.9%、97%、97.1%、97.2%、97.3%、97.4%、97.41%、97.42%、97.43%、97.44%、97.45%、97.46%、97.47%、97.48%、97.49%、97.5%、97.51%、97.52%、97.53%、97.54%、97.55%、97.56%、97.57%、97.58%、97.59%、97.6%、97.61%、97.62%、97.63%、97.64%、97.65%、97.66%、97.67%、97.68%、97.69%97.7%、97.71%、97.72%、97.73%、97.74%、97.75%、97.76%、97.77%、97.78%、97.79%、97.8%、97.81%、97.82%、97.83%、97.84%、97.85%、97.86%、97.87%、97.88%、97.89%、97.9%、97.91%、97.92%、97.93%、97.94%、97.95%、97.96%、97.97%、97.98%、97.99%、98%、98.01%、98.02%、98.03%、98.04%、98.05%、98.06%、98.07%、98.08%、98.09%、98.1%、98.11%、98.12%、98.13%、98.14%、98.15%、98.16%、98.17%、98.18%、98.19%、98.2%、98.21%、98.22%、98.23%、98.24%、98.25%、98.26%、98.27%、98.28%、98.29%、98.3%、98.31%、98.32%、98.33%、98.34%、98.35%、98.36%、98.37%、98.38%、98.39%、98.4%、98.41%、98.42%、98.43%、98.44%、98.45%、98.46%、98.47%、98.48%、98.49%,98.5%、98.51%、98.52%、98.53%、98.54%、98.55%、98.56%、98.57%、98.58%、98.59%、98.6%、98.61%、98.62%、98.63%、98.64%、98.65%、98.66%、98.67%、98.68%、98.69%、98.7%、98.71%、98.72%、98.73%、98.74%、98.75%、98.76%、98.77%、98.78%、98.79%、98.8%、98.81%、98.82%、98.83%、98.84%、98.85%、98.86%、98.87%、98.88%、98.89%、98.9%、98.91%、98.92%、98.93%、98.94%、98.95%、98.96%、98.97%、98.98%、98.99%、99%、99.01%、99.02%、99.03%、99.04%、99.05%、99.06%、99.07%、99.08%、99.09%、99.1%、99.11%、99.12%、99.13%、99.14%、99.15%、99.16%、99.17%、99.18%、99.19%、99.2%、99.21%、99.22%、99.23%、99.24%、99.25%、99.26%、99.27%、99.28%、99.29%、99.3%、99.31%、99.32%、99.33%、99.34%、99.35%、99.36%、99.37%、99.38%、99.39%、99.4%、99.41%、99.42%、99.43%、99.44%、99.45%、99.46%、99.47%、99.48%、99.49%、99.5%、99.51%、99.52%、99.53%、99.54%、99.55%、99.56%、99.57%、99.58%、99.59%、99.6%,99.61%、99.62%、99.63%、99.64%、99.65%、99.66%、99.67%、99.68%、99.69%、99.7%、99.71%、99.72%、99.73%、99.74%、99.75%、99.76%、99.77%、99.78%、99.79%、99.8%、99.81%、99.82%、99.83%99.84%、99.85%、99.86%、99.87%、99.88%、99.89%、99.9%、99.91%、99.92%、99.93%、99.94%、99.95%、99.96%、99.97%、99.98%和99.99%的序列同一性。如本文使用的,特别理解术语“与SEQ ID NO:X的序列同一性”或“相同的SEQ ID NO:X”分别等价于术语“在SEQ ID NO:X的长度上与SEQ ID NO:X的序列的序列同一性”或“在SEQ ID NO:X的长度上与SEQ ID NO:X的序列相同”。

适用于本发明的优选刺突多肽是具有PDCoV刺突SEQ ID NO:4、8、12、18或28中所示序列的多肽和/或与SEQ ID NO:4、8、12、18、28、31、35、47和/或53具有至少80%的同源性的PEDV刺突SEQ ID NO:31、35,47或53,例如与SEQ ID NO:4、8、12、18、28、31、35、47和/或53具有至少85%的同源性,例如与SEQ ID NO:4、8、12、18、28、31、35、47和/或53具有至少85%的同源性,例如与SEQ ID NO:4、8、12、18、28、31、35、47和/或53具有至少90%的同源性,例如与SEQ ID NO:4、8、12、18、28、31、35、47和/或53具有至少95%、至少98%或至少99%的同源性。

术语“疫苗”和“免疫原性组合物”在本文中以广义进行定义,以指能够在用疫苗接种的动物中刺激免疫应答的可施用形式的任何类型的生物制剂。疫苗一般而言可以基于病毒本身(例如,杀死/灭活的或减毒的)或病毒的免疫原性(抗原性)组分。在本发明的一个实施方案中,疫苗(免疫原性组合物)优选包括以杀死/灭活形式的病毒剂或作为亚单位疫苗呈递的病毒的抗原部分。在本文中,当涉及免疫原性组合物使用时,术语“临床症状的减少”指与所讨论的疾病或状况相关的任何症状的减轻或改善(或部分或完全)。因此,通过本免疫原性组合物减少由PDCoV和/或PEDV感染的猪的临床症状一般导致病毒脱落和/或与由PDCoV和/或PEDV感染相关的一种或多种临床症状的减少(例如,小于10日龄的猪中的急性水样腹泻、急性呕吐、脱水、厌食、嗜睡、抑郁和高死亡率)。同样地,通过本免疫原性组合物减少由PDCoV和/或PEDV感染的猪的临床症状一般导致病毒脱落和/或与由PDCoV和/或PEDV感染相关的一种或多种临床症状的减少(例如,小于10日龄的猪中的急性水样腹泻、急性呕吐、脱水、厌食、嗜睡、抑郁和高死亡率)

本领域技术人员将理解,本文使用的组合物可以掺入已知的可注射、生理学上可接受的无菌溶液内。为了制备用于肠胃外注射或输注的即用型溶液,水性等渗溶液例如盐水或血浆蛋白质溶液可容易获得。另外,本发明的免疫原性和疫苗组合物可以包括兽医学上可接受的载体、稀释剂、等渗剂、稳定剂或佐剂。

本发明的方法包括但不限于在对象中引发针对单独或与彼此组合或其它共感染的PDCoV感染和/或PEDV感染的免疫应答的方法,其包括给对象施用免疫原性组合物的步骤,所述免疫原性组合物包含灭活/杀死的PDCoV和/或PEDV、减毒的PDCoV和/或PEDV、和/或衍生自PDCoV和/或PEDV的重组刺突抗原。优选地,引发针对超过一种血清型、或PDCoV或PEDV的毒株的免疫应答。本发明的组合物可以用于预防PDCoV感染、PEDV感染和/或PEDV/PDCoV共感染。优选地,此类免疫应答减少与一种或多种PDCoV基因型和/或PEDV基因型/基因组感染相关或由其引起的一种或多种临床体征的发生率或严重性。

在本文中,可以对其施用本发明的组合物的合适对象和有需要的对象包括需要预防性治疗病毒相关感染、疾病或状况的动物。其中通过使用本发明的组合物或方法刺激免疫应答的动物包括家畜例如猪、牛、家禽(例如鸡、鸭、鹅或火鸡)、山羊和绵羊,以及驯养动物例如小鼠、兔子、犬、猫和马。优选的动物包括猪科动物、鼠科动物、马科动物、兔类动物和牛科动物。最优选地,在猪中刺激免疫应答。

本发明还提供了减少与PDCoV感染和/或PEDV感染相关或由其引起的一种或多种临床体征的发生率或严重性的方法,所述方法包括施用本发明的免疫原性组合物的步骤,所述免疫原性组合物包含灭活/杀死的PDCoV疫苗、灭活/杀死的PEDV疫苗、和/或与衍生自如本文提供的PDCoV和/或PEDV的重组刺突抗原组合,且优选载体分子,使得PDCoV和/或PEDV感染的临床体重的发生率或严重性相对于未接受如本文提供的免疫原性组合物的对象减少至少10%,优选至少20%,甚至更优选至少30%,甚至更优选至少50%,甚至更优选至少70%,最优选100%。此类临床特征包括水样腹泻、呕吐和脱水。这些临床症状中的任一种都可以起因于由具有2a的基因群或者任何其它PEDV基因群包括G1a、G1b或G2b的PEDV的感染。同样地,这些临床体征中的任一种都可以起因于由不同基因型的PDCoV的感染和/或共感染。

在一个实施方案中,本免疫原性组合物包括化学灭活形式的PEDV。包含化学灭活的PDCoV(SEQ ID NO:1或2、5或6、9或10)病毒的疫苗是特别期望的。另外,本免疫原性组合物可以包括化学灭活形式的PEDV。包含化学灭活的PEDV(SEQ ID NO:29或32或SEQ ID NO:33或36)病毒的疫苗是特别期望的。本领域技术人员已知的各种化学灭活剂可以用于灭活病毒。乙烯亚胺和相关衍生物,例如二乙烯亚胺(“BEI”)和乙酰乙烯亚胺,是用于灭活SDCo和/或PED病毒的合适化学灭活剂的实例。其它化学灭活剂,例如β-丙内酯或醛(例如甲醛和戊二醛)也可以用于灭活病毒。

本免疫原性组合物和/或疫苗一般包括期望地可以具有生物粘附特性的佐剂,特别是在病毒被设计为能够鼻内施用的情况下。合适佐剂的实例包括交联的烯属不饱和羧酸聚合物,例如交联的丙烯酸聚合物。如本文使用的,术语“交联的丙烯酸聚合物”指由单体混合物形成的聚合物和共聚物,所述单体混合物包括丙烯酸作为混合物中的占优势单体。合适的交联丙烯酸聚合物的实例包括以商品名934P和971(可从B.F.Goodrich Co.,Cleveland,Ohio获得)商购可得的那些。用于本疫苗中的一种特别合适的佐剂是其布氏粘度不超过约20,000cPs的交联的丙烯酸聚合物,(如在20rpm下测量为以pH 7.5的1.0重量%水溶液)。当需要生物粘附性佐剂时,利用如在两片新鲜切除的兔胃组织之间测量的具有至少约50达因/cm2的生物粘附特性的佐剂可能是有利的(如通过美国专利号4,615,697中所述的程序测定的)。

本发明还涉及免疫对象的方法,其包括向对象施用如本文所述的任何免疫原性组合物。

术语“免疫”涉及通过向待免疫的对象施用免疫原性组合物的主动免疫,从而引起针对此类免疫原性组合物中包括的抗原的免疫学应答。

优选地,免疫导致畜群中单独或与PEDV感染组合的特定PDCoV感染的发生率降低,或导致由特定PDCoV和/或PEDV感染引起或与其相关的临床体征的严重性减少。

此外,用如本文提供的免疫原性组合物免疫有需要的对象,导致预防对象被PDCoV和/或PEDV感染的感染。甚至更优选地,免疫导致针对PDCoV和/或PEDV感染的有效、持久的免疫应答。应理解,所述时间段将持续超过2个月,优选超过3个月,更优选超过4个月,更优选超过5个月,更优选超过6个月。应理解,免疫接种可能并非在所有免疫的对象中是有效的。

优选地,在该上下文设想一群对象,其通常(即没有免疫接种)将发展通常由PDCoV感染和/或PEDV感染引起或与其相关的临床体征。本领域技术人员可以毫不费力地确定畜群的对象是否得到有效免疫。优选地,如果与未免疫或用在本发明之前可获得的免疫原性组合物免疫,但随后被特定的PDCoV和/或PEDV感染的对象相比,在至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少80%、至少90%,再更优选至少95%且最优选100%的给定畜群的对象中的临床体征在发生率或严重性方面减少至少10%,更优选至少20%,再更优选至少30%,甚至更优选至少40%,再更优选至少50%,甚至更优选至少60%,再更优选至少70%,甚至更优选至少80%,再更优选至少90%,再更优选至少95%,且最优选100%,则免疫应该是有效的。

用于预防在有需要的对象中由PDCoV和/或PEDV引起的临床体征的方法,或保护猪不受与PDCoV和/或PEDV相关的疾病的方法,包括给猪施用含有灭活/杀死的PDCoV、和/或灭活/杀死的PEDV、和/或衍生自PDCoV和/或PEDV的刺突抗原的免疫原性组合物和/或疫苗。疫苗可以使用各种方法施用,包括鼻内、经口和/或肠胃外(例如,肌内)施用。在该方法的一个实施方案中,例如,含有灭活的PDCoV和/或灭活的PEDV的免疫原性组合物肌内施用一次或多次(例如,以2–4周的间隔)。在该方法的另一个实施方案中,例如,含有灭活的PDCoV和/或灭活的PEDV的疫苗经口施用一次或多次(例如,以2–4周的间隔)。在一个替代实施方案中,经口施用可以随后为肌内施用疫苗至少一次和/或在其之前(例如,在疫苗的肠胃外施用之后和/或之前2–4周)。理想地,给定畜群中的所有猪都按规定的间隔接种疫苗,以便保护防止疾病症状的传播。

还提供了产生灭活/杀死的PDCoV疫苗、灭活/杀死的PEDV疫苗、或组合PDCoV/PEDV免疫原性组合物的方法。对于PDCoV的生产,该方法通常包括用PDCo病毒,例如用PDCo病毒SEQ ID NO:1或2、5或6和9或10接种猪睾丸细胞。接种的猪睾丸细胞一般至少温育直到CPE为止,然后从温育的细胞中收获PED病毒。收获的含病毒的流体可以用化学灭活剂如二乙烯亚胺处理,以形成灭活/杀死的PDCo病毒。通常,灭活的病毒例如通过浓缩且与其它组分共混进一步加工,以产生商业制剂。例如,含有灭活病毒的流体可以浓缩,并且与佐剂和/或关于一种或多种其它猪病原体的抗原共混。为了产生PEDV,该方法通常包括用PED病毒,例如用PED病毒SEQ ID NO:29或SEQ ID NO:33接种猿猴细胞。使接种的猿猴细胞一般至少温育直至观察到CPE,然后从温育的细胞中收获PED病毒。收获的含病毒的流体可以用化学灭活剂如二乙烯亚胺处理,以形成灭活/杀死的PED病毒。通常,灭活的病毒例如通过浓缩且与其它组分共混进一步加工,以产生商业制剂。例如,含有灭活病毒的流体可以浓缩,并且与佐剂和/或关于一种或多种其它猪病原体的抗原共混。

还提供了产生经由表达PDCoV和/或PEDV刺突蛋白的重组杆状病毒在昆虫细胞中生成的重组表达的刺突抗原疫苗的方法。在一个示例性实施方案中的方法包括将PDCoV刺突编码序列(例如,SEQ ID NO:3、7、11、17或27)和/或PEDV刺突编码序列(SEQ ID NO:30、34、46或52),包括例如PDCoV刺突-Fc抗原构建体(SEQ ID NO:17)、和/或PDCoV刺突Baculodisplay构建体(SEQ ID NO:27)、和/或PEDV 2a刺突构建体(SEQ ID NO:46)、和/或PEDV 2b刺突构建体(SEQ ID NO:52),克隆到杆状病毒载体内,并且共转染Sf9昆虫细胞。

本申请还涉及试剂盒,其包括组合的(1)能够给猪施用疫苗的分配器;以及(2)化学灭活的PDCoV、和/或化学灭活的PEDV、和/或衍生自含有PDCoV和/或PEDV的疫苗的重组刺突抗原,其能够保护不受与PDCoV和/或PEDV相关的疾病。该试剂盒可以包括能够将其内容物分配为小滴例如气溶胶、雾化喷雾和/或液滴的分配器,以及例如当鼻内和/或肌内施用时,能够保护不受与PDCoV和/或PEDV相关的疾病的疫苗形式。

在本申请自始至终,该文本涉及本组合物和/或相关方法的各种实施方案。所描述的各种实施方案意欲提供各种说明性实施例,并且不应解释为对替代种类的描述。相反,应该注意,本文提供的各种实施方案的描述可以具有重叠的范围。本文讨论的实施方案仅是说明性的,并不意欲限制本发明的范围。

附图说明

下述附图构成本说明书的部分,并且被包括以进一步证实本发明的某些方面。通过参考与本文呈现的具体实施方案的详细描述组合的这些附图中的一个或多个,可以更好地理解本发明。

图1关于PDCoV分离株/毒株的完全基因组核苷酸邻近树。比例尺指示p距离。GenBank登录号连同两个字母的州或三个字母的国家代码一起列出(CHN–中国,TJK–泰国,LAO–老挝)。

图2关于PDCoV分离株/毒株的刺突氨基酸邻近树。比例尺指示p距离。GenBank登录号连同两个字母的州或三个字母的国家代码一起列出(CHN–中国,TJK–泰国,LAO–老挝)。

图3A PDCoV S1-IgG2a Fc的PDCoV S1-IgG2a Fc杆状病毒-蛋白质图的示意图。

图3B PDCoVS BD的PDCoVS BD Baculodisplay杆状病毒-蛋白质图的示意图。

图4与安慰剂(第3组)相比,用PDCoV原型(第1、2和4组,分别为BaculoS-BD-PDCoV、全细胞PDCoV和BaculoS-FC-PDCoV)的血清学研究。

图5与安慰剂(第3组)相比,如通过基于S1-IgG2a-Fc的ELISA检测的,在用PDCoV原型接种疫苗后的血清学应答:(第1、2和4组,分别为BaculoS-BD-PDCoV、全细胞PDCoV和BaculoS-FC-PDCoV)。

图6A PEDV 2a BD Baculodisplay杆状病毒蛋白质图的示意图。

图6B PEDV 2b BD Baculodisplay杆状病毒-蛋白质图的示意图。

图7在PEDV BD攻击后,通过组和天数的生存性。

图8接种疫苗的动物中的IgA水平。

具体实施方式

本发明提供了免疫原性组合物,其包括灭活/杀死形式的PDCoV和/或PEDV和/或重组表达的PDCoV和/或PEDV-刺突抗原。免疫原性组合物被设计用于保护猪不受与PDCoV和/或PEDV相关的疾病。免疫原性组合物通常包括化学灭活形式的PDCoV和/或PEDV,并且包括化学灭活/杀死的PDCoV和/或PEDV病毒的那些是特别期望的。在另一个实施方案中,免疫原性组合物包括例如经由表达PDCoV和/或PEDV刺突蛋白的重组杆状病毒,在昆虫细胞中生成的重组表达的PDCoV和/或PEDV刺突抗原。

一般地,本发明提供了包含一种或多种猪丁型冠状病毒(PDCoV)抗原和佐剂的免疫原性组合物,其中在猪丁型冠状病毒(PDCoV)中的是任何PDCoV:a.)其由SEQ ID NO:1、5或9编码,和/或包含SEQ ID NO:1、5或9的序列,和/或包含SEQ ID NO:1、5或9的RNA等价物;b.)其序列与SEQ ID NO:1、5或9至少99%相同,和/或与SEQ ID NO:1、5或9的RNA等价物至少99%相同,c.)其刺突蛋白由SEQ ID NO:3、7或11的核酸序列编码;d.)其刺突蛋白由与SEQ ID NO:3、7或11至少90%相同的核酸序列编码;e.)其由SEQ ID NO:2、6或10编码;或f.)其序列与SEQ ID NO:2、6或10至少99%相同。

在一个具体方面,根据上述句子的免疫原性组合物,其中佐剂是水包油型乳状液。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组合物是重组抗原或灭活的完整猪丁型冠状病毒PDCoV抗原。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述抗原是灭活的完整PDCoV抗原。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述PDCoV抗原是化学灭活的。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述PDCoV抗原通过用化学灭活剂处理而化学灭活,所述化学灭活剂包括选自乙烯亚胺、二乙烯亚胺、乙酰乙烯亚胺及其混合物的化合物。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述PDCoV通过用二乙烯亚胺处理而化学灭活。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述佐剂是基于水包油型乳状液的佐剂。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中灭活的猪丁型冠状病毒(PDCoV)包含SEQ ID NO:1、5或9,和/或包含SEQ ID NO:1、5或9的RNA等价物。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述PDCoV抗原是重组抗原。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述重组抗原包含选自以下的一种或多种免疫原性组分:a.)编码猪丁型冠状病毒(PDCoV)刺突蛋白的抗原的分离核酸,其中所述重组刺突多肽与SEQ ID NO:4、8、12、18或28具有至少90%的同源性;b.)包含a)的分离核酸的重组载体;c.)由a)的核酸编码的重组PDCoV刺突蛋白;和d.)其任何组合。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中此类免疫原性组合物包含药学上可接受的载体和/或赋形剂。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述水包油型乳状液是基于水包油型乳状液的佐剂。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组合物进一步包含一种或多种另外的抗原。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中另外的抗原是猪流行性腹泻病毒(PEDV)的抗原,其中所述猪流行性腹泻病毒(PEDV)是任何PEDV:a.)其由SEQ ID NO:29或33编码,和/或包含SEQ ID NO:29或33的序列,和/或包含SEQ ID NO:29或33的RNA等价物;b.)其序列与SEQ ID NO:29或33至少99%相同,和/或与SEQ ID NO:29或33的RNA等价物至少99%相同;c.)其刺突蛋白由SEQ ID NO:30、34、46或52的核酸序列编码;d.)其刺突蛋白由与SEQ ID NO:30、34、46或52至少90%相同的核酸序列编码;e.)其由SEQ ID NO:32或36编码;或f.)其由与SEQ ID NO:32或36至少99%相同的序列编码。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述另外的PEDV抗原是重组抗原或灭活的完整猪流行性腹泻病毒(PEDV)。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述抗原是灭活的完整猪流行性腹泻病毒(PEDV)。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述抗原是重组PEDV抗原。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述重组抗原包含选自以下的一种或多种免疫原性组分:a.)编码猪流行性腹泻病毒(PEDV)刺突蛋白的抗原的分离核酸,其中所述抗原与SEQ ID NO:31、35、47或53具有至少90%的同源性;b.)包含a)的分离核酸的重组载体;c.)由a)的核酸编码的重组PEDV刺突蛋白;和d.)其任何组合。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述重组抗原包含选自以下的一种或多种免疫原性组分:猪流行性腹泻病毒(PEDV)的结构蛋白M、E或N。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组分是所述分离的核酸。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组分是所述重组载体。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组分是所述重组猪流行性腹泻病毒(PEDV)刺突蛋白。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其中所述免疫原性组分是组合。

本发明进一步涉及用于减少与猪丁型冠状病毒(PDCoV)相关的疾病的临床症状的方法,其包括给猪施用如上述句子中任一中所述的免疫原性组合物。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其包括给猪施用包含灭活的完整PDCoV抗原的免疫原性组合物。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其包括给猪施用包含PDCoV抗原作为重组抗原的免疫原性组合物。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中此类施用的免疫原性组合物包含选自以下的一种或多种免疫原性组分:a.)其由SEQ ID NO:1编码,和/或包含SEQ ID NO:1、5或9的序列,和/或包含SEQ ID NO:1、5或9的RNA等价物;b.)其序列与SEQ ID NO:1、5或9至少99%相同,和/或与SEQ ID NO:1、5或9的RNA等价物至少99%相同;c.)其刺突蛋白由SEQ ID NO:3、7、11、17、27的核酸序列编码;d.)其刺突蛋白由与SEQID NO:3、7、11、17或27至少90%相同的核酸序列编码;e.)其由SEQ ID NO:2、6或10编码;和f.)其序列与SEQ ID NO:2、6或10至少99%相同。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中此类施用的免疫原性组合物包含选自以下的一种或多种免疫原性组分:a.)其由SEQ ID NO:1、5或9的序列编码,或者包含SEQ ID NO:1、5或9的序列;b.)其序列与SEQ ID NO:1、5或9至少99%相同;d.)其刺突蛋白由SEQ ID NO:3、7、11、17或27的核酸序列编码;和e.)其刺突蛋白由与SEQ ID NO:3、7、11、17或27至少90%相同的核酸序列编码。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中此类施用的免疫原性组合物包含重组抗原,所述重组抗原包含选自以下的一种或多种免疫原性组分:a.)编码猪流行性腹泻病毒、猪丁型冠状病毒(PDCoV)刺突蛋白抗原的分离核酸,其中所述重组刺突多肽与SEQ ID NO:4、8、12、18或28具有至少90%的同源性;b.)包含a)的分离核酸的重组载体;c.)由a)的核酸编码的重组猪丁型冠状病毒(PDCoV)刺突蛋白;和d.)其任何组合。

本发明进一步涉及用于减少与猪丁型冠状病毒(PDCoV)和猪流行性腹泻病毒(PEDV)相关的疾病的临床症状的方法,其包括给猪施用如上述句子中任一中所述的任何免疫原性组合物,优选地,其中所述免疫原性组合物包含一种或多种PDCoV抗原和一种或多种另外的抗原,其中所述另外的抗原是猪流行性腹泻病毒(PEDV)的抗原。

在本发明的一个进一步的具体方面,该方法如上述句子中所述,其包括给此类猪施用免疫原性组合物,其中所述另外的PEDV抗原是重组抗原或灭活的完整猪流行性腹泻病毒(PEDV)。

在本发明的一个进一步的具体方面,该方法如上述句子中所述,其包括给此类猪施用如上述句子中任一中所述的免疫原性组合物。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性组合物的分配器;和b.)如上述句子中任一中所述的免疫原性组合物。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性的分配器;和b.)如上述句子中任一中所述的免疫原性组合物,更具体而言,其中所述抗原是灭活的完整PDCoV抗原,甚至更具体而言,其中所述PDCoV抗原是化学灭活的;更具体而言,其中所述PDCoV抗原通过用化学灭活剂处理而化学灭活,所述化学灭活剂包括选自乙烯亚胺、二乙烯亚胺、乙酰乙烯亚胺及其混合物的化合物;优选地,其中所述PDCoV通过用二乙烯亚胺处理而化学灭活。更具体而言,其中所述佐剂是基于水包油型乳状液的佐剂;且甚至更优选地,其中所述灭活的猪丁型冠状病毒(PDCoV)包含SEQ ID NO:1、5或9,和/或包含SEQ ID NO:1、5或9的RNA等价物。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性组合物的分配器;和b.)如上述句子中任一中所述的免疫原性组合物,更具体而言,其中所述PDCoV抗原是重组抗原,甚至更具体而言,其中所述重组抗原包含选自以下的一种或多种免疫原性组分:a.)编码猪丁型冠状病毒(PDCoV)刺突蛋白抗原的分离核酸,其中所述重组刺突多肽与SEQ ID NO:4、8、12、18或28具有至少90%的同源性;b.)包含a)的分离核酸的重组载体;c.)由a)的核酸编码的重组PDCoV刺突蛋白;和d)其任何组合,甚至更具体而言,其中此类免疫原性组合物包含药学上可接受的载体和/或赋形剂;并且甚至更具体而言,其中所述水包油型乳状液是基于水包油型乳状液的佐剂。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)和猪流行性腹泻病毒(PEDV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性组合物的分配器;和b.)如上述句子中任一中所述的免疫原性组合物,具体地其中所述另外的抗原是猪流行性腹泻病毒(PEDV),并且是如上述句子中任一中所述的任何PEDV抗原。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)和猪流行性腹泻病毒(PEDV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性组合物的分配器;和b.)如上述句子中任一中所述的免疫原性组合物,具体地其中所述另外的抗原是猪流行性腹泻病毒(PEDV),是如上述句子中任一中所述的任何重组PEDV抗原或任何灭活的完整猪流行性腹泻病毒(PEDV)。

本发明进一步涉及用于在猪中诱导针对与猪丁型冠状病毒(PDCoV)和猪流行性腹泻病毒(PEDV)相关的疾病的免疫原性应答的试剂盒,其包含:a.)能够给猪施用免疫原性组合物的分配器;和b.)如上述句子中任一中所述的免疫原性组合物,具体地其中所述另外的抗原是如上述句子中任一中所述的任何重组PEDV抗原。

本发明进一步涉及制备如上述句子中任一中所述的猪丁型冠状病毒(PDCoV)免疫原性组合物的方法,其进一步包括:a.)用猪丁型冠状病毒(PDCoV)接种猪睾丸细胞;b.)温育接种的猪睾丸细胞;c.)从温育的细胞中收获猪丁型冠状病毒(PDCoV);以及d.)用化学灭活剂,优选用选自乙烯亚胺、二乙烯亚胺、乙酰乙烯亚胺或其混合物的化合物处理收获的细胞,以形成灭活的猪丁型冠状病毒(PDCoV)抗原。

在本发明的一个进一步的具体方面,该方法如上述句子中所述,其中所述猪丁型冠状病毒(PDCoV)包含另外的特征:a.)由SEQ ID NO:1、5或9的序列编码,或者包含SEQ IDNO:1、5或9的序列,b.)与SEQ ID NO:1、5或9至少99%相同的序列,c.)由SEQ ID NO:3、7、11、17或27的核酸序列编码的刺突蛋白;d.)由与SEQ ID NO:3、7、11、17或27至少90%相同的核酸序列编码的刺突蛋白。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中所述猪丁型冠状病毒(PDCoV)包含另外的特征SEQ ID NO:1、5或9,和/或SEQ ID NO:1、5或9的RNA等价物。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中所述猪睾丸细胞是AI-ST细胞。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中所述化学灭活剂包括二乙烯亚胺。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其进一步包括给猪丁型冠状病毒(PDCoV)抗原添加基于水包油型乳状液的佐剂的另外特征。

本发明进一步涉及用于制备免疫原性组合物的方法,所述免疫原性组合物包含如前述句子中任一中所述的重组猪丁型冠状病毒(PDCoV)抗原,所述方法包括:a.)在宿主细胞中表达猪丁型冠状病毒(PDCoV)的抗原,b.)收获猪丁型冠状病毒(PDCoV)细胞的抗原;以及c)将基于水包油型乳状液的佐剂加入步骤b)的猪丁型冠状病毒(PDCoV)抗原中。

在本发明的一个进一步的具体方面,该方法如上述句子中所述,其中所述猪丁型冠状病毒(PDCoV)抗原包含:a.)编码猪丁型冠状病毒(PDCoV)刺突蛋白的抗原的分离核酸,其中所述重组刺突多肽与SEQ ID NO:3、7、11、17或27具有至少90%的同源性;b.)包含a)的分离核酸的重组载体;c.)由a)的核酸编码的重组猪丁型冠状病毒(PDCoV)刺突蛋白;和d.)其任何组合。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中所述猪丁型冠状病毒(PDCoV)的抗原由重组杆状病毒载体表达。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中所述猪丁型冠状病毒(PDCoV)的抗原在昆虫细胞中表达。

在本发明的一个进一步的具体方面,该方法如上述句子中任一中所述,其中作为另外的特征,基于水包油的佐剂是基于水包油型乳状液的佐剂。

本发明进一步涉及包含如上述句子中任一中所述的PDCoV的免疫原性组合物,其用于减少与猪丁型冠状病毒(PDCoV)相关的疾病的症状。

本发明进一步涉及包含PDCoV和至少一种另外的抗原的免疫原性组合物,其中至少一种另外的抗原是猪流行性腹泻病毒(PEDV)的抗原,其用于减少与猪丁型冠状病毒(PDCoV)和/或猪流行病毒(PEDV)相关的疾病的症状。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其进一步包含PEDV基因型2a的一种或多种抗原,具体地PEDV具有北美起源。

在本发明的一个进一步的具体方面,免疫原性组合物如上述句子中任一中所述,其进一步包含一种或多种PDCoV抗原,其中所述PDCoV具有北美起源。

更一般地说,本领域技术人员已知的各种化学灭活剂可以用于灭活病毒。乙烯亚胺和相关衍生物,例如二乙烯亚胺(BEI)和乙酰乙烯亚胺,是用于灭活PED病毒的合适化学灭活剂的实例。其它化学灭活剂,例如β-丙内酯、醛(例如甲醛)和/或去污剂(例如,去污剂、 X或烷基三甲基铵盐)也可以用于灭活病毒。可以使用本领域技术人员已知的标准方法进行灭活。可以以定期时间间隔获取样品并且测定残留的活病毒。用适当的特异性单克隆或多克隆抗体监测对适当细胞系的致细胞病变作用和/或荧光染色可以用于检测残留活病毒的存在。

用BEI的灭活可以通过将储备BEI溶液(例如,通过将0.1–0.2M 2-溴-乙胺氢溴酸盐加入0.1–0.2N水性NaOH中形成的溶液)与病毒流体组合至约1–5mM BEI的最终浓度来实现。通常通过将BEI-病毒混合物保持在35–40℃(例如,37℃)下,伴随24–72小时的持续混合来进行灭活。通过加入硫代硫酸钠溶液至超过BEI浓度的最终浓度(例如,加入以BEI体积17%的硫代硫酸钠以中和过量的BEI),随后为混合,可以终止病毒灭活。

本免疫原性组合物通常包括佐剂,以及需要时的一种或多种乳化剂,例如去污剂,其掺入到灭活/杀死的PEDV。合适的佐剂包括例如乙酸维生素E增溶物、氢氧化铝、磷酸铝或氧化铝、(矿物)油乳状液、非离子型去污剂、角鲨烯和皂苷。可以使用的其它佐剂包括油基佐剂,例如弗氏完全佐剂(FCA)和弗氏不完全佐剂(FIA)。已发现交联的烯属不饱和羧酸聚合物,例如971聚合物,是用于本灭活的PEDV免疫原性组合物中的特别适合的佐剂。

关于合适的水包油型乳状液的实例是基于的佐剂,例如(水包油型乳状液)、(具有二甲基二十八烷基溴化铵(DDA)的水包油型)、(具有专利权免疫刺激剂的水包油型)、(具有交联聚合物的由水包油型组成的双重佐剂)、以及(不含动物起源组分的水包油型乳状液。(MVP Technologies,Inc.Omaha,Nebr.,USA)。包含灭活的PEDV或重组PEDV蛋白的药物/疫苗组合物已用水包油型乳状液有效地佐剂化,优选用此类基于的佐剂,更优选用(不含动物起源组分的水包油型乳状液)和(不含动物起源组分的水包油型乳状液)。

一般有利的是以剂量单位形式配制本组合物以便于施用和剂量的均匀性。如本文使用的,剂量单位形式指适合作为待治疗的哺乳动物对象的单位剂量的物理上离散的单位;每个单位含有预定数量的活性材料,其计算为与所需的药物载体结合产生所需的疗效。关于灭活/杀死的PDCoV和/或PEDV、和/或重组表达的PDCoV和/或PEDV抗原的剂量单位形式的规格由其它因素决定且取决于其它因素:(a)活性材料的独特特征和待达到的特定疗效;(b)复合此类活性材料用于治疗疾病的领域中固有的局限性;以及(c)剂量单位形式的预期施用方式。

通常配制主要活性成分,用于以有效量与如本文公开的以剂量单位形式的合适的药学上可接受的载体方便有效的施用。单位剂型可以例如含有PDCoV和/或PEDV抗原,其量范围为1至约5个相对效力单位(“RPU”)。该抗原量一般以约1至约25/ml的载体存在。在含有补充活性成分的组合物的情况下,剂量通过参考补充活性成分的通常剂量和施用方式来确定。

本发明的疫苗通常包括用药学上可接受的载体配制的灭活的PDCoV和/或PEDV。适合于注射使用的药物形式通常包括无菌水溶液(在水溶性的情况下)或分散体,以及用于临时制备无菌可注射溶液或分散体的无菌粉末。该制剂应期望地是无菌的且流动至存在容易可注射性的程度。剂型在制造和贮存条件下应该是稳定的,并且通常针对微生物如细菌和真菌的污染作用进行防腐。载体可以是溶剂或分散介质,其含有例如水、乙醇、多元醇(例如甘油、丙二醇、液体聚乙二醇等等)、其合适的混合物和植物油。一种可能的载体是生理盐溶液。例如,通过使用涂层如卵磷脂、在分散体的情况下通过维持所需的粒度和通过使用表面活性剂,可以维持溶液的适当流动性。可以通过各种抗菌剂和抗真菌剂,例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、山梨酸、硫柳汞(乙基汞-硫代水杨酸钠)、新霉素(deomycin)、庆大霉素等等,来达到微生物作用的预防。在许多情况下,可能优选包括等渗剂,例如糖或氯化钠。需要时,可以通过在组合物中使用延迟吸收的试剂,例如单硬脂酸铝和明胶,来达到可注射组合物的延长吸收。

可以通过将灭活的病毒以所需的量,根据需要与如上文列举的各种其它成分一起掺入适当的溶剂中,随后为过滤灭菌来制备无菌可注射溶液。一般地,可以通过将各种活性成分掺入无菌媒介物内来制备分散体,所述无菌媒介物含有基础分散介质和来自上文列举的那些的所需其它成分。在用于制备无菌可注射溶液的无菌粉末的情况下,优选的制备方法是真空干燥和冷冻干燥技术,所述技术从其先前无菌过滤的溶液产生活性成分加上任何其它所需成分的粉末。

向本组合物中加入稳定剂以改善灭活病毒的稳定性也可能是有利的。合适的稳定剂包括例如甘油/EDTA、碳水化合物(如山梨糖醇、甘露醇、海藻糖、淀粉、蔗糖、葡聚糖或葡萄糖)、蛋白质(如白蛋白或酪蛋白)和蛋白质降解产物(如部分水解的明胶)。需要时,可以通过本领域已知的方法来缓冲制剂,使用试剂例如碱金属磷酸盐,例如磷酸氢二钠、磷酸二氢钠、磷酸氢钾和/或磷酸二氢钾。其它溶剂例如乙醇或丙二醇,可以用于增加成分在疫苗制剂中的溶解度和/或溶液的稳定性。可以用于本制剂中的进一步添加剂包括常规抗氧化剂和常规螯合剂,如乙二胺四乙酸(EDTA)。

本发明的组合物和方法可以通过下述实施例进行说明,所述实施例呈现以说明本发明,并且帮助教导普通技术人员如何制备且使用其。这些实施例决不预期缩小或以其它方式限制本发明的范围。

除非另有说明,否则本发明的实践将采用分子生物学、微生物学、重组DNA技术、蛋白质化学和免疫学的常规技术,所述技术在本领域的技术内。此类技术在文献中充分解释。参见例如Sambrook,Fritsch&Maniatis,Molecular Cloning:A Laboratory Manual,第I、II和III卷,第二版(1989);DNA Cloning,第I和II卷(D.N.Glover编辑,1985);Oligonucleotide Synthesis(M.J.Gait编辑1984);Nucleic Acid Hybridization(B.D.Hames&S.J.Higgins编辑1984);Animal Cell Culture(R.K.Freshney编辑1986);Immobilized Cells and Enzymes(IRL press,1986);Perbal,B.,A Practical Guide toMolecular Cloning(1984);该系列,Methods In Enzymology(S.Colowick和N.Kaplan编辑,Academic Press,Inc.);Protein purification methods–a practical approach(E.L.V.Harris和S.Angal,编辑,IRL Press at Oxford University Press);和Handbookof Experimental Immunology,第I-IV卷(D.M.Weir和C.C.Blackwell编辑,1986,Blackwell Scientific Publications)。

应理解,本发明并不限于特定的DNA、RNA、多肽序列或工艺参数,因为这些当然可以改变。还应理解,本文使用的术语仅用于描述本发明的特定实施方案的目的,并不预期是限制性的。必须注意,如在本说明书和所附权利要求中使用的,单数形式“一个”、“一种”和“该/所述”包括复数指示物,除非内容另有明确说明。因此,例如,提及“抗原”包括两种或更多种抗原的混合物;提及“赋形剂”包括两种或更多种赋形剂的混合物等等。

定义:

除非另有定义,否则本文使用的所有技术和科学术语具有与本申请在提交时所属领域的技术人员通常理解相同的含义。术语的含义和范围应该是明确的;然而,在任何潜在歧义的情况下,本文提供的定义优先于任何字典或外部定义。此外,除非上下文另有要求,否则单数术语应该包括复数,并且复数术语应该包括单数。在本文中,除非另有说明,否则“或”的使用意指“和/或”。此外,术语“包括(including)”以及其它形式如“包括(includes)”和“包括(included)”的使用并非限制性的。本文提及的所有专利和出版物都引入本文作为参考。

“针对疾病的保护”、“保护性免疫”、“功能性免疫”和类似短语,意指针对通过施用本发明的一种或多种治疗组合物或其组合生成的疾病或状况的应答,其导致比已暴露于疾病或感染的非免疫对象中预期的更少的有害效应。即,感染的有害效应的严重性在接种疫苗的对象中减轻。在接种疫苗的对象中可以减少、减缓或可能完全预防感染。在本文中,当意欲完全预防感染时,它被具体陈述。如果未陈述完全预防,则该术语包括部分预防。

在本文中,“临床体征的发生率和/或严重性的减少”或“临床症状的减少”意指但不限于与野生型感染相比,减少组中受感染对象的数目、减少或消除显示出感染的临床体征的对象数目、或减少一个或多个对象中存在的任何临床体征的严重性。例如,它应该指病原体负荷的减少、病原体脱落、病原体传播中的减少、或PEDV症状的任何临床体征的减少。优选地,与未接受组合物并且变得被感染的对象相比,接受本发明的治疗组合物的一个或多个对象中这些临床体征减少至少10%。更优选地,在接受本发明的组合物的对象中,临床体征减少至少20%,优选至少30%,更优选至少40%,且甚至更优选至少50%。

术语“增加的保护”在本文中意指但不限于,在接种疫苗的对象组相对于未接种疫苗的对照对象组中,与被传染剂(优选PDCoV和/或PEDV)的感染相关的一种或多种临床症状的统计学显著减少。术语“临床症状的统计学显著减少”意指但不限于,在用传染剂攻击后,与未接种疫苗的对照组相比,接种疫苗的对象组中的至少一种临床症状的发生率的频率低至少10%,优选20%,更优选30%,甚至更优选50%,且甚至更优选70%。

“持久保护”应该指“改善的功效”,其持续至少3周,但更优选至少3个月,再更优选至少6个月。在家畜的情况下,最优选的是持久保护应该持续直到动物在其下上市用于肉类的平均年龄。

“免疫原性或免疫学组合物”指包含至少一种猪丁型冠状病毒和/或猪流行性腹泻病毒和/或其免疫原性部分的物质组合物,其在宿主中引发细胞或抗体介导的针对组合物的免疫学应答。在本发明的一个优选实施方案中,免疫原性组合物诱导免疫应答,并且更优选地,赋予针对PDCoV和/或PEDV感染的一种或多种临床体征的保护性免疫。

如本文使用的,免疫原性组合物还指包含本文所述的任何PDCoV和/或PEDV刺突多肽的组合物。根据进一步的实施方案,此类免疫原性组合物进一步包含表达所述PDCoV和/或PEDV刺突蛋白的病毒载体,优选重组杆状病毒的至少一部分。此外,免疫原性组合物可以包含i)上述任何PDCoV或PEDV蛋白,优选以上述浓度,ii)表达所述PDCoV和/或PEDV刺突蛋白的病毒载体,优选重组杆状病毒的至少一部分,以及iii)细胞培养上清液的一部分。

因此,根据一个方面,本发明涉及用于减少猪群中的PDCoV和/或PEDV感染百分比的方法,其包括给所述猪施用有效量的PDCoV和/或PEDV刺突抗原或包含PDCoV和/或PEDV抗原的免疫原性组合物的步骤,其中所述PDCoV和/或PEDV抗原是重组PDCoV和/或PEDV刺突抗原,优选杆状病毒表达的PDCoV和/或PEDV刺突蛋白。优选地,那些重组或杆状病毒表达的PDCoV和/或PEDV刺突具有如本文所述的序列。

“免疫应答”或“免疫学应答”意指但不限于,针对目的组合物或疫苗的细胞和/或抗体介导的免疫应答的发展。通常,免疫应答或免疫学应答包括但不限于下述效应中的一种或多种:抗体、B细胞、辅助性T细胞、抑制性T细胞和/或细胞毒性T细胞的产生或活化,特别针对目的组合物或疫苗中包括的一种或多种抗原。优选地,宿主将展示治疗或保护性免疫(记忆)反应,使得增强针对新感染的抗性和/或减少疾病的临床严重性。此类保护可以通过减少症状数目、症状严重性、或缺乏与病原体感染相关的一种或多种症状、延迟病毒血症发作、减少病毒持续时间、减少总体病毒负荷和/或减少病毒***得到证实。

抗原的“免疫保护量”或“免疫有效量”或“产生免疫应答的有效量”是有效诱导受体中的免疫原性应答的量。免疫原性应答可能足以用于诊断目的或其它测试,或者可能足以预防由疾病因子感染引起的疾病的体征或症状,包括不良健康效应或其并发症。可以诱导体液免疫或细胞介导的免疫或两者。例如,间接地通过测量抗体滴度、淋巴细胞增殖测定,或直接地通过监测用野生型毒株攻击后的体征和症状,可以评估动物对免疫原性组合物的免疫原性应答,而由疫苗赋予的保护性免疫可以通过测量例如临床体征中的减少来评估,所述临床体征例如对象的死亡率、发病率、温度数、总体身体状况以及总体健康和表现。免疫应答可以包括但不限于细胞和/或体液免疫的诱导。“免疫原性”意指诱发免疫或抗原应答。因此,免疫原性组合物是诱导免疫应答的任何组合物。

“治疗有效量”指在接受抗原或疫苗的对象中诱导免疫应答的抗原或疫苗的量,其足以预防或减少由被病原体如病毒或细菌感染引起的疾病的临床体征或症状,包括不利的健康影响或其并发症。可以诱导体液免疫或细胞介导的免疫或体液和细胞介导的免疫两者。例如,间接地通过测量抗体滴度、淋巴细胞增殖测定,或直接地通过监测用野生型毒株攻击后的体征和症状,可以评估动物对疫苗的免疫原性应答。由疫苗赋予的保护性免疫可以通过测量例如临床体征中的减少来评估,所述临床体征例如对象的死亡率、发病率、温度数、总体身体状况以及总体健康和表现。治疗有效的疫苗的量可以根据使用的特定佐剂、使用的特定抗原或对象的状况而变化,并且可以由本领域技术人员确定。

如本文使用的,“药学或兽医学上可接受的载体”包括任何和所有溶剂、分散介质、包衣、佐剂、稳定剂、稀释剂、防腐剂、抗菌剂和抗真菌剂、等渗剂、吸附延迟剂等等。在一些优选的实施方案且尤其是包括冻干的免疫原性组合物的那些中,用于本发明中的稳定剂包括用于冻干或冷冻干燥的稳定剂。

在一些实施方案中,本发明的免疫原性组合物含有佐剂。如本文使用的,“佐剂”可以包括氢氧化铝和磷酸铝、皂苷例如Quil A、QS-21(Cambridge Biotech Inc.,CambridgeMA)、GPI-0100(Galenica Pharmaceuticals,Inc.,Birmingham,AL)、油包水型乳状液、水包油型乳状液、水包油包水型乳状液。

乳状液可以特别基于轻质液体石蜡油(欧洲药典型);类异戊二烯油如角鲨烷或角鲨烯;起因于烯烃,特别是异丁烯或癸烯的低聚反应的油;含有线性烷基的酸或醇的酯,更特别是植物油、油酸乙酯、丙二醇二-(辛酸酯/癸酸酯)、甘油基三-(辛酸酯/癸酸酯)或丙二醇二油酸酯;分支脂肪酸或醇的酯,特别是异硬脂酸酯。该油与乳化剂组合使用以形成乳状液。乳化剂优选为非离子表面活性剂,特别是脱水山梨糖醇、二缩甘露醇(例如脱水甘露醇油酸酯)、二醇、聚甘油、丙二醇和油酸、异硬脂酸、蓖麻油酸或羟基硬脂酸(其是任选乙氧基化的)的酯和聚氧丙烯-聚氧乙烯共聚物嵌段,特别是Pluronic产品,尤其是L121。参见Hunter等人The Theory and Practical Application of Adjuvants(Ed.Stewart-Tull,D.E.S.),JohnWiley和Sons,NY,第51-94页(1995)和Todd等人Vaccine 15:564-570(1997)。示例性佐剂是在由M.Powell和M.Newman,Plenum Press,1995编辑的“Vaccine Design,TheSubunit and Adjuvant Approach”的第147页上描述的SPT乳状液,以及在同一本书的第183页上描述的乳状液MF59。

关于合适的水包油型乳状液的实例是基于的佐剂,例如(水包油型乳状液o/w)、(具有二甲基二十八烷基溴化铵(DDA)的水包油型(o/w))、(具有专利权免疫刺激剂的水包油型(o/w))、(具有交联聚合物的由水包油型(o/w)组成的双重佐剂)、以及(不含动物起源组分的水包油型乳状液)。(MVP Technologies,Inc.Omaha,Nebr.,USA)。包含灭活的PDCoV、灭活的PEDV和/或重组的PDCoV和/或PEDV蛋白的药物/疫苗组合物已用水包油型乳状液,优选用此类基于的佐剂,更优选用(水包油型乳状液o/w)和/或(不含动物起源组分的水包油型乳状液)有效地佐剂化。

用于兽医疫苗中的合适的吸附剂氢氧化铝凝胶的实例包括 REHYDRAGEL-LV;REHYDRAGEL-HPA;REHYDRAPHOS(GeneralChemical,Berkeley Heights,New Jersey,USA)。

佐剂的进一步实例是选自丙烯酸或甲基丙烯酸的聚合物和马来酸酐与烯基衍生物的共聚物的化合物。有利的佐剂化合物是尤其与糖或多元醇的聚烯基醚交联的丙烯酸或甲基丙烯酸的聚合物。这些化合物通过术语卡波姆(Phameuropa第8卷,No.2,1996年6月)已知。本领域技术人员还可以参考美国专利2,909,462,其描述了与具有至少3个羟基,优选不超过8个羟基的多羟基化的化合物交联的此类丙烯酸聚合物,至少三个羟基的氢原子被替换为具有至少2个碳原子的不饱和脂族原子团。优选的原子团是含有2至4个碳原子的那些,例如,乙烯基、烯丙基和其它烯属不饱和基团。不饱和原子团本身可以含有其它取代基,例如甲基。以名称CARBOPOL.RTM.销售的产品(也称为聚丙烯酸);(BF Goodrich,Ohio,USA)是特别合适的。它们与烯丙基蔗糖或烯丙基季戊四醇交联。其中,可以提到CARBOPOL.RTM.974P(也称为聚丙烯酸)、CARBOPOL.RTM.934P(也称为聚丙烯酸)和CARBOPOL.RTM.971P(也称为聚丙烯酸)。最优选的是使用CARBOPOL.RTM.971P(也称为聚丙烯酸)。在马来酸酐和链烯基衍生物的共聚物中,共聚物EMA(Monsanto)是马来酸酐和乙烯的共聚物。这些聚合物在水中的溶解导致酸溶液被中和,优选至生理学pH,以便得到免疫原性、免疫学或疫苗组合物本身掺入其内的佐剂溶液。

进一步合适的佐剂尤其包括但不限于RIBI佐剂系统(Ribi Inc.)、嵌段共聚物(CytRx,Atlanta Ga.)、SAF-M(Chiron,Emeryville Calif.)、单磷酰脂质A、阿夫立定脂质-胺佐剂、来自大肠杆菌(E.coli)的热不稳定性肠毒素(重组或其它)、霍乱毒素或胞壁酰二肽。

预期佐剂可以以每剂量约100μg至约10mg的量,优选以每剂量约100μg至约10mg的量,更优选以每剂量约500μg至约5mg的量,甚至更优选以每剂量约750μg至约2.5mg的量,且最优选以每剂量约1mg的量加入。可替代地,佐剂可以以按最终产物的体积计约0.01至50%的浓度,优选以约2%至30%的浓度,更优选以约5%至25%的浓度,再更优选以约7%至22%的浓度,且最优选以10%至20%的浓度。

“稀释剂”可以包括水、盐水、右旋糖、乙醇、甘油等等。等渗剂可以尤其包括氯化钠、右旋糖、甘露醇、山梨糖醇和乳糖。稳定剂尤其包括白蛋白和乙二胺四乙酸的碱金属盐等。

“分离的”意指“通过人为”从其天然状态改变,即,如果它在自然界中出现,则它已从其原始环境改变或取出、或两者。例如,天然存在于活生物中的多核苷酸或多肽不是“分离的”,但与其天然状态的共存材料分开的相同多核苷酸或多肽是“分离的”,如该术语在本文中采用的。

“安全性”指在疫苗接种后,在接种疫苗的动物中不存在不良后果,包括但不限于:基于病毒的疫苗可能回复毒力、临床上显著的副作用如持续的全身性疾病或在疫苗施用部位处无法接受的炎症。

如本文使用的,术语“疫苗接种”或“接种疫苗”或其变体意指但不限于,包括施用本发明的免疫原性组合物的过程,当施用于动物时,所述免疫原性组合物在动物中引发、或者能够直接或间接地引发针对PDCoV和/或PEDV的免疫应答。

在本发明的上下文中,“死亡率”指通过PDCoV感染、PEDV感染和/或由PDCoV和PEDV的共感染引起的死亡,并且包括其中感染如此严重使得动物被实施安乐死以预防痛苦并且对其生命提供人道结局的情况。

在本文中,“有效剂量”意指但不限于引发或能够引发免疫应答的抗原量,所述免疫应答在抗原施用于其的动物中产生临床症状的减少。

如本领域已知的,“序列同一性”指两个或更多个多肽序列或者两个或更多个多核苷酸序列,即参考序列和待与参考序列比较的给定序列之间的关系。通过在序列已最佳比对以产生最高程度的序列相似性之后,将给定序列与参考序列进行比较来确定序列同一性,如通过此类序列的串之间的匹配所确定的。在此类比对后,在逐个位置的基础上确定序列同一性,例如,如果在该位置处,核苷酸或氨基酸残基是相同的,则序列在特定位置处是“相同的”。然后将此类位置同一性的总数目除以参考序列中的核苷酸或残基的总数目,以得到%序列同一性。序列同一性可以通过已知方法容易地计算,所述方法包括但不限于以下中描述的那些:Computational Molecular Biology,Lesk,A.N.,编辑,OxfordUniversity Press,New York(1988),Biocomputing:Informatics and Genome Projects,Smith,D.W.,编辑,Academic Press,New York(1993);Computer Analysis of SequenceData,Part I,Griffin,A.M.和Griffin,H.G.,编辑,Humana Press,New Jersey(1994);Sequence Analysis in Molecular Biology,von Heinge,G.,Academic Press(1987);Sequence Analysis Primer,Gribskov,M.和Devereux,J.,编辑,M.Stockton Press,NewYork(1991);以及Carillo,H.和Lipman,D.,SIAM J.Applied Math.,48:1073(1988),其教导引入本文作为参考。确定序列同一性的优选方法设计为给出测试序列之间的最大匹配。确定序列同一性的方法在可公开获得的计算机程序中编码,所述程序确定给定序列之间的序列同一性。此类程序的实例包括但不限于GCG程序包(Devereux,J.等人Nucleic AcidsResearch,12(1):387(1984))、BLASTP、BLASTN和FASTA(Altschul,S.F.等人J.Molec.Biol.,215:403-410(1990)。BLAST程序可从NCBI和其它来源公开获得(BLASTManual,Altschul,S.等人NCVI NLM NIH Bethesda,MD 20894,Altschul,S.F.等人J.Molec.Biol.,215:403-410(1990),其教导引入本文作为参考)。这些程序使用缺省缺口权重最佳地比对序列,以便产生给定序列和参考序列之间的最高水平的序列同一性。作为说明,通过具有与参考核苷酸序列具有至少例如85%,优选90%,甚至更优选95%“序列同一性”的核苷酸序列的多核苷酸,预期给定多核苷酸的核苷酸序列与参考序列相同,除了给定多核苷酸序列可以包括参考核苷酸序列的至多15个,优选至多10个,甚至更优选至多5个点突变/100个核苷酸之外。换言之,在具有相对于参考核苷酸序列具有至少85%,优选90%,甚至更优选95%同一性的核苷酸序列的多核苷酸中,参考序列中至多15%,优选10%,甚至更优选5%的核苷酸可以被缺失或用另一种核苷酸取代,或者可以将参考序列中的总核苷酸的至多15%,优选10%,甚至更优选5%的多个核苷酸***参考序列内。参考序列的这些突变可以出现在参考核苷酸序列的5'或3'末端位置处或者这些末端位置之间的任何位置,个别散布在参考序列中的核苷酸中或者参考序列内的一个或多个邻接组中。类似地,通过具有与参考氨基酸序列具有至少例如85%,优选90%,甚至更优选95%的序列同一性的给定氨基酸序列的多肽,预期多肽的给定氨基酸序列与参考序列相同,除了给定的多肽序列可以包括参考氨基酸序列的至多15个,优选至多10个,甚至更优选至多5个氨基酸改变/各100个氨基酸之外。换言之,为了获得与参考氨基酸序列具有至少85%,优选90%,甚至更优选95%的序列同一性的给定多肽序列,参考序列中至多15%,优选至多10%,甚至更优选至多5%的氨基酸残基可以被缺失或用另一种氨基酸取代,或者可以将参考序列中的氨基酸残基总数目的至多15%,优选至多10%,甚至更优选至多5%的多个氨基酸***参考序列内。参考序列的这些改变可以出现在参考氨基酸序列的氨基或羧基末端位置处或者这些末端位置之间的任何位置,个别散布在参考序列中的残基中或者参考序列内的一个或多个邻接组中。优选地,并不相同的残基位置因保守氨基酸取代而不同。然而,在确定序列同源性时,保守取代不包括作为匹配。

“保守取代”指氨基酸残基由具有相似特征或特性(包括大小、疏水性等)的另一种氨基酸残基的取代,使得总体功能性不显著改变。

如本文使用的,“序列同源性”指确定扣除保守取代的两个序列的相关性的方法。为了确定序列同源性,最佳比对两个或更多个序列,并且在需要时引入缺口。换言之,为了获得与参考序列具有95%序列同源性的多肽,参考序列中85%,优选90%,甚至更优选95%的氨基酸残基必须与另一种氨基酸匹配或包含由另一种氨基酸的保守取代,或者参考序列中至多15%,优选至多10%,甚至更优选至多5%的总氨基酸残基的多个氨基酸(不包括保守取代的氨基酸)可以***参考序列内。优选地,同源序列包含至少50个,甚至更优选100个,甚至更优选250个,甚至更优选500个氨基酸的段。

术语“序列同一性”或“百分比同一性”在本文中可互换使用。为了本发明的目的,此处定义为了确定两个氨基酸序列或两个核酸序列的百分比同一性,将序列比对用于最佳比较目的(例如,可以在第一氨基酸或核酸的序列中引入缺口,用于与第二氨基酸或核酸序列的最佳比对)。然后比较在相应的氨基酸或核苷酸位置处的氨基酸或核苷酸残基。当第一序列中的位置被与第二序列中的相应位置相同的氨基酸或核苷酸残基占据时,则该分子在该位置处是相同的。两个序列之间的百分比同一性是由序列共享的相同位置数目的函数(即,%同一性=相同位置的数目/位置(即重叠位置)总数目x 100)。优选地,两个序列具有相同的长度。当在提及蛋白质中使用序列同一性时,认识到并非相同的残基位置经常因保守氨基酸取代而不同,其中氨基酸残基取代具有相似化学特性(例如电荷或疏水性)的其它氨基酸残基,并且因此不改变分子的功能特性。当序列在保守取代方面不同时,可以向上调整百分比序列同一性,以校正取代的保守性质。通过此类保守取代而不同的序列被称为具有“序列相似性”或“相似性”。

可以在待比较的两个序列的整个长度上或在两个序列的片段上进行序列比较。通常,比较将在待比较的两个序列的全长上进行。然而,序列同一性可以在例如二十、五十、一百或更多个邻接氨基酸残基的区域上进行。

本领域技术人员将意识到几种不同的计算机程序可用于确定两个序列之间的同源性的事实。例如,可以使用数学算法完成序列的比较和两个序列之间的百分比同一性的确定。在一个优选的实施方案中,使用Needleman和Wunsch(J.Mol.Biol.(48):444-453(1970))算法确定两个氨基酸或核酸序列之间的百分比同一性,所述算法已掺入GAP程序内的Accelrys GCG软件包中(可在http://www.accelrys.com/products/gcg/处获得),使用Blosum 62矩阵或PAM250矩阵,以及16、14、12、10、8、6或4的缺口权重和1、2、3、4、5或6的长度权重。技术人员将了解,使用不同的算法时,所有这些不同的参数将产生略微不同的结果,但两个序列的总体百分比同一性并不显著改变。

本发明的蛋白质序列或核酸序列可以进一步用作“查询序列”,以针对公共数据库进行搜索,以例如鉴定其它家族成员或相关序列。可以使用Altschul等人(1990)J.Mol.Biol.215:403-10的BLASTN和BLASTP程序(版本2.0)进行此类搜索。BLAST蛋白质搜索可以用BLASTP程序、得分=50、字长=3进行,以获得与本发明的蛋白质分子同源的氨基酸序列。为了获得用于比较目的的加缺口的比对,可以利用如Altschul等人(1997)NucleicAcids Res.25(17):3389-3402中所述的Gapped BLAST。当利用BLAST和Gapped BLAST程序时,可以使用分别程序(例如,BLASTP和BLASTN)的缺省参数。参见在http://www.ncbi.nlm.nih.gov/处的美国国家生物技术信息中心(National Center forBiotechnology Information)主页。

本发明的请求保护的PEDV还应该涵盖PEDV分离株1251-125-10(“125-10”)的变体及其亚片段的变体。此类变体具有与Oklahoma毒株(SEQ ID NO 29和34)的特征基本相同的免疫学特性。术语“具有基本上相同的免疫学特性”涵盖(但不限于)所述变体基本上有效地治疗或预防由如下所述的PEDV引起的临床体征或者改善如下所述的功效参数。

本发明的请求保护的PDCoV还应该涵盖PDCoV分离株NSVL、PDCoV 2.0307和PDCoV5.0327的变体及其亚片段的变体。此类变体具有与NSVL毒株(SEQ ID NO:1和2),以及分离株PDCoV 2.0307(SEQ ID NO:5和6)和PDCoV 5.0327(SEQ ID NO:9和10)的特征基本上相同的免疫学特性。术语“具有基本上相同的免疫学特性”涵盖(但不限于)所述变体在治疗或预防由如本文所述的PDCoV引起的临床体征或者改善如本文所述的功效参数方面基本上有效。除了可以用于疫苗中的各种PDCoV毒株外,重组刺突蛋白抗原,包括其亚片段,也可以用于疫苗中。同样地,示例性刺突蛋白序列包括但不限于具有与下文列出的PDCoV分离株或‘变体’基本上相同的免疫学特性的那些。

关于PDCoV序列(SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12,17、18、27和28)或PEDV序列SEQ ID NO:29、30、31、32、33、34、35、36、46、47、52和53)(例如,多肽或核酸序列)的术语“变体”预期意指基本上相似的序列。对于包含开放读码框的核苷酸序列,变体包括由于遗传密码的简并性而编码天然蛋白的相同氨基酸序列的那些序列。变体核苷酸序列还包括合成衍生的核苷酸序列,例如通过使用定点诱变和开放读码框生成的那些,编码天然蛋白质,以及编码相对于天然蛋白质具有氨基酸取代用于密码子优化目的的多肽的那些。一般地,使用标准参数,使用描述的比对程序之一,与参考序列相比较,本发明的核苷酸序列变体将具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%或89%,更优选至少90%、91%、92%、93%或94%,且最优选至少95%、96%、96.1%、96.2%、96.3%、96.4%、96.5%、96.6%、96.7%、96.8%、96.9%、97%、97.1%、97.2%、97.3%、97.4%、97.41%、97.42%、97.43%、97.44%、97.45%、97.46%、97.47%、97.48%、97.49%、97.5%、97.51%、97.52%、97.53%、97.54%、97.55%、97.56%、97.57%、97.58%、97.59%、97.6%、97.61%、97.62%、97.63%、97.64%、97.65%、97.66%、97.67%、97.68%、97.69%97.7%、97.71%、97.72%、97.73%、97.74%、97.75%、97.76%、97.77%、97.78%、97.79%、97.8%、97.81%、97.82%、97.83%、97.84%、97.85%、97.86%、97.87%、97.88%、97.89%、97.9%、97.91%、97.92%、97.93%、97.94%、97.95%、97.96%、97.97%、97.98%、97.99%、98%、98.01%、98.02%、98.03%、98.04%、98.05%、98.06%、98.07%、98.08%、98.09%、98.1%、98.11%、98.12%、98.13%、98.14%、98.15%、98.16%、98.17%、98.18%、98.19%、98.2%、98.21%、98.22%、98.23%、98.24%、98.25%、98.26%、98.27%、98.28%、98.29%、98.3%、98.31%、98.32%、98.33%、98.34%、98.35%、98.36%、98.37%、98.38%、98.39%、98.4%、98.41%、98.42%、98.43%、98.44%、98.45%、98.46%、98.47%、98.48%、98.49%,98.5%、98.51%、98.52%、98.53%、98.54%、98.55%、98.56%、98.57%、98.58%、98.59%、98.6%、98.61%、98.62%、98.63%、98.64%、98.65%、98.66%、98.67%、98.68%、98.69%、98.7%、98.71%、98.72%、98.73%、98.74%、98.75%、98.76%、98.77%、98.78%、98.79%、98.8%、98.81%、98.82%、98.83%、98.84%、98.85%、98.86%、98.87%、98.88%、98.89%、98.9%、98.91%、98.92%、98.93%、98.94%、98.95%、98.96%、98.97%、98.98%、98.99%、99%、99.01%、99.02%、99.03%、99.04%、99.05%、99.06%、99.07%、99.08%、99.09%、99.1%、99.11%、99.12%、99.13%、99.14%、99.15%、99.16%、99.17%、99.18%、99.19%、99.2%、99.21%、99.22%、99.23%、99.24%、99.25%、99.26%、99.27%、99.28%、99.29%、99.3%、99.31%、99.32%、99.33%、99.34%、99.35%、99.36%、99.37%、99.38%、99.39%、99.4%、99.41%、99.42%、99.43%、99.44%、99.45%、99.46%、99.47%、99.48%、99.49%、99.5%、99.51%、99.52%、99.53%、99.54%、99.55%、99.56%、99.57%、99.58%、99.59%、99.6%,99.61%、99.62%、99.63%、99.64%、99.65%、99.66%、99.67%、99.68%、99.69%、99.7%、99.71%、99.72%、99.73%、99.74%、99.75%、99.76%、99.77%、99.78%、99.79%、99.8%、99.81%、99.82%、99.83%99.84%、99.85%、99.86%、99.87%、99.88%、99.89%、99.9%、99.91%、99.92%、99.93%、99.94%、99.95%、99.96%、99.97%、99.98%和99.99%的序列同一性。

关于PDCoV序列(SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12,17、18、27和28)(例如,多肽或核酸序列)的术语“变体”预期意指基本上相似的序列。因此,本发明的免疫原性组合物可以有效地掺入所有公认的PDCoV毒株或分离株,优选包括但不必限于与以下具有至少约90%总体核苷酸同一性的所有毒株:保藏为GenBank登录号KP981395.1的分离株NSVL-SCDV(猪丁型冠状病毒毒株USA/IL/2014/026PDV_P11);保藏为GenBank登录号KP757890的分离株CHN-AH-2004;保藏为GenBank登录号KP757891的分离株CHN-HB-2014;保藏为GenBank登录号:KP757892的分离株CHN-JS-2014;保藏为GenBank登录号KR131621的分离株PDCoV/CHJXNI2/2015;保藏为GenBank登录号KR150443的毒株USA/Arkansas61/2015;保藏为GenBank登录号KR265847的毒株USA/Minnesota442/2014;保藏为GenBank登录号KR265848的毒株USA/Minnesota214/2014;保藏为GenBank登录号KR265849的毒株USA/Michigan447/2014;保藏为GenBank登录号KR265850的毒株USA/Michigan448/2014;保藏为GenBank登录号KR265851的毒株USA/Indiana453/2014;保藏为GenBank登录号KR265852的毒株USA/Illinois449/2014;保藏为GenBank登录号KR265853的毒株USA/Minnesota/2013;保藏为GenBank登录号KR265854的毒株USA/Minnesota454/2014;保藏为GenBank登录号KR265855的毒株USA/Minnesota455/2014;保藏为GenBank登录号KR265856的毒株USA/Illinois272/2014;保藏为GenBank登录号KR265857的毒株USA/Illinois273/2014;保藏为GenBank登录号KR265858的毒株USA/NorthCarolina452/2014;保藏为GenBank登录号KR265859的毒株USA/Minnesota159/2014;保藏为GenBank登录号KR265860的毒株USA/Nebraska209/2014;保藏为GenBank登录号KR265861的毒株USA/Nebraska210/2014;保藏为GenBank登录号KR265862的毒株USA/Ohio444/2014;保藏为GenBank登录号KR265863的毒株USA/Ohio445/2014;保藏为GenBank登录号KR265864的毒株USA/Minnesota292/2014;保藏为GenBank登录号KR265865的毒株USA/Iowa459/2014;保藏为GenBank登录号KT021234的毒株CH/SXD1/2015;保藏为GenBank登录号KT266822的毒株CH/Sichuan/S27/2012;保藏为GenBank登录号KT336560的分离株CHN-HN-2014;保藏为GenBank登录号KU051641的毒株PDCoV/Swine/Thailand/S5011/2015;保藏为GenBank登录号KU051649的毒株PDCoV/Swine/Thailand/S5015L/2015;保藏为GenBank登录号KU981059的毒株NH;保藏为GenBank登录号KU981060的毒株NH分离株第0代;保藏为GenBank登录号KU981061的NH分离株第5代;保藏为GenBank登录号KU981062的毒株NH分离株第10代;保藏为GenBank登录号KU984334的分离株TT_1115;保藏为GenBank登录号KX022602的毒株PDCoV/USA/Iowa136/2015;保藏为GenBank登录号KX022603的毒株PDCoV/USA/Minnesota140/2015;保藏为GenBank登录号KX022604的毒株PDCoV/USA/Nebraska137/2015;保藏为GenBank登录号KX022605的毒株PDCoV/USA/Nebraska145/2015;以及保藏为GenBank登录号KX118627的分离株P1_16_BTL_0115/PDCoV/2016/Lao。

如本领域已知的,术语“基因群”指属内的相关病毒;其可以进一步细分成遗传聚类。鉴定的PEDV基因群包括包含亚群G1a、G1b、R(减毒/适应)的群G1;以及包含亚群G2a和G2b的G2。G2a基因群的成员包括共享几个独特的核苷酸变化的中国毒株AH2012(GenBank登录号:KC210145)和北美毒株。分别地,毒株MN和IA2与AH2012具有99.6%的核苷酸同一性,并且毒株IA1与AH2012具有99.5%的核苷酸同一性。研究人员已推测,AH2012样病毒可能传播到中国东部地区,然后运输至美国,并且很可能是最接近北美株的祖先。基因群2a的成员与基因群1a的原型PEDV毒株CV777仅共享大约96.9%的相似性(Bridgen等人1993;Huang等人2013;GenBank:AF353511.1)。像这样,基于历史CV777衍生的G1a毒株或DR13衍生的G1b毒株的减毒PEDV疫苗可以与新近出现的中国和北美G2a PEDV毒株在抗原上较不相关。

密切相关的北美PEDV分离株US/Colorado/2013(GenBank登录号:KF272920.1)也已通过Marthaler等人,2013得到报道。如同上述北美分离株,CO/13的完全PEDV基因组与GenBank中可获得的其它完全PEDV基因组具有96.5%至99.5%的核苷酸同一性,与中国毒株AH2012(GenBank登录号KC210145)具有最高的核苷酸同一性(99.5%)。中国毒株株AH2012是2a基因群的成员。北美分离株CO/13的完全基因组与PEDV参考毒株CV777的那种的比较,显示CO/13含有1个核苷酸的***(在位置48处)和5'UTR中5个核苷酸的缺失(在位置73和83至86处),而刺突基因含有16个核苷酸的***(位置20804、20810至20820、20843和21053至21055)和7个核苷酸的缺失(位置20853和21118至21124)。

已出现了PEDV的其它变体,称为'INDEL'毒株,其与较老的原型毒株相比经常是天然减毒的。这些也可以用作其中病毒是活减毒的或灭活的疫苗。在此类情况下,可能仅需要最低限度的进一步传代来提供安全的疫苗减毒。因此,本发明的示例性疫苗病毒还包括对于刺突蛋白或基于完整的病毒序列,与此类毒株具有至少95%、96%、96.1%、96.2%、96.3%、96.4%、96.5%、96.6%、96.7%、96.8%、96.9%、97%、97.1%、97.2%、97.3%、97.4%、97.41%、97.42%、97.43%、97.44%、97.45%、97.46%、97.47%、97.48%、97.49%、97.5%、97.51%、97.52%、97.53%、97.54%、97.55%、97.56%、97.57%、97.58%、97.59%、97.6%、97.61%、97.62%、97.63%、97.64%、97.65%、97.66%、97.67%、97.68%、97.69%97.7%、97.71%、97.72%、97.73%、97.74%、97.75%、97.76%、97.77%、97.78%、97.79%、97.8%、97.81%、97.82%、97.83%、97.84%、97.85%、97.86%、97.87%、97.88%、97.89%、97.9%、97.91%、97.92%、97.93%、97.94%、97.95%、97.96%、97.97%、97.98%、97.99%、98%、98.01%、98.02%、98.03%、98.04%、98.05%、98.06%、98.07%、98.08%、98.09%、98.1%、98.11%、98.12%、98.13%、98.14%、98.15%、98.16%、98.17%、98.18%、98.19%、98.2%、98.21%、98.22%、98.23%、98.24%、98.25%、98.26%、98.27%、98.28%、98.29%、98.3%、98.31%、98.32%、98.33%、98.34%、98.35%、98.36%、98.37%、98.38%、98.39%、98.4%、98.41%、98.42%、98.43%、98.44%、98.45%、98.46%、98.47%、98.48%、98.49%,98.5%、98.51%、98.52%、98.53%、98.54%、98.55%、98.56%、98.57%、98.58%、98.59%、98.6%、98.61%、98.62%、98.63%、98.64%、98.65%、98.66%、98.67%、98.68%、98.69%、98.7%、98.71%、98.72%、98.73%、98.74%、98.75%、98.76%、98.77%、98.78%、98.79%、98.8%、98.81%、98.82%、98.83%、98.84%、98.85%、98.86%、98.87%、98.88%、98.89%、98.9%、98.91%、98.92%、98.93%、98.94%、98.95%、98.96%、98.97%、98.98%、98.99%、99%、99.01%、99.02%、99.03%、99.04%、99.05%、99.06%、99.07%、99.08%、99.09%、99.1%、99.11%、99.12%、99.13%、99.14%、99.15%、99.16%、99.17%、99.18%、99.19%、99.2%、99.21%、99.22%、99.23%、99.24%、99.25%、99.26%、99.27%、99.28%、99.29%、99.3%、99.31%、99.32%、99.33%、99.34%、99.35%、99.36%、99.37%、99.38%、99.39%、99.4%、99.41%、99.42%、99.43%、99.44%、99.45%、99.46%、99.47%、99.48%、99.49%、99.5%、99.51%、99.52%、99.53%、99.54%、99.55%、99.56%、99.57%、99.58%、99.59%、99.6%,99.61%、99.62%、99.63%、99.64%、99.65%、99.66%、99.67%、99.68%、99.69%、99.7%、99.71%、99.72%、99.73%、99.74%、99.75%、99.76%、99.77%、99.78%、99.79%、99.8%、99.81%、99.82%、99.83%99.84%、99.85%、99.86%、99.87%、99.88%、99.89%、99.9%、99.91%、99.92%、99.93%、99.94%、99.95%、99.96%、99.97%、99.98%和99.99%的序列同一性或更高序列同一性的那些,无论是测量氨基酸还是编码核苷酸序列。

术语“北美起源的PEDV”意指包含SEQ ID NO:29和/或33和/或SEQ ID NO:32和/或36的PEDV分离株,和/或与SEQ ID NO:29和/或33具有至少99%序列同一性的任何PEDV分离株,和/或与SEQ ID.NO:29和/或33的RNA等价物至少99%相同的PEDV分离株,和/或其中刺突蛋白由SEQ ID NO:30、34、46或52编码的PEDV分离株,和/或其中刺突蛋白与SEQ ID:30、34、46或52具有至少98%的序列同一性的PEDV分离株,和/或其中表达的刺突蛋白与SEQ IDNO:31、35、47、53具有至少90%同源性的任何PEDV分离株。

如本领域已知的,术语“进化枝”指由祖先及其所有后代组成的组,系统发育树中的单个“分支”。作为一个例子,祖先可以是个体、种群或物种。基因群可以包括多个进化枝,例如AH2012在与北美分离株不同的进化枝中。

根据一个进一步的实施方案,本发明还涉及包含如本文所述的任何此类核酸分子的载体。换言之,本发明涉及载体,其包括任何此类刺突、M、E、N PEDV蛋白或其部分的编码序列。优选地,所述载体是表达载体,其允许表达任何此类此类刺突、M、E和/或N PEDV蛋白或蛋白质的部分。根据本发明的载体是适合于在体外或体内转染或感染细菌、酵母或动物细胞的那些。

用于制备和/或使用用于表达的载体(或重组体)的载体和方法可以通过以下中公开的方法或类似于以下中公开的方法:美国专利4,603,112、4,769,330、5,174,993、5,505,941、5,338,683、5,494,807、4,722,848、5,942,235、5,364,773、5,762,938、5,770,212、5,942,235、382,425,PCT公开WO 94/16716、WO 96/39491、WO 95/30018;Paoletti,"Applications of pox virus vectors to vaccination:An update,"PNAS USA 93:11349-11353,October 1996;Moss,"Genetically engineered poxviruses forrecombinant gene expression,vaccination,and safety,"PNAS USA 93:11341-11348,October 1996;Smith等人,美国专利号4,745,051(重组杆状病毒);Richardson,C.D.(编辑),Methods in Molecular Biology 39,"Baculovirus Expression Protocols"(1995Humana Press Inc.);Smith等人,"Production of Human Beta Interferon inInsect Cells Infected with a Baculovirus Expression Vector",Molecular andCellular Biology,December,1983,第3卷,No.12,第2156-2165页;Pennock等人,"Strongand Regulated Expression of Escherichia coli B-Galactosidase in Infect Cellswith a Baculovirus vector,"Molecular and Cellular Biology March 1984,第4卷,No.3,第406页;EPA0 370 573;于1986年10月16日提交的美国申请号920,197;EP专利公开号265785;美国专利号4,769,331(重组疱疹病毒);Roizman,"The function of herpessimplex virus genes:A primer for genetic engineering of novel vectors,"PNASUSA 93:11307-11312,October 1996;Andreansky等人,"The application ofgenetically engineered herpes simplex viruses to the treatment ofexperimental brain tumors,"PNAS USA 93:11313-11318,October 1996;Robertson等人,"Epstein-Barr virus vectors for gene delivery to B lymphocytes",PNAS USA93:11334-11340,October 1996;Frolov等人,"Alphavirus-based expression vectors:Strategies and applications,"PNAS USA 93:11371-11377,October 1996;Kitson等人,J.Virol.65,3068-3075,1991;美国专利号5,591,439、5,552,143;WO 98/00166;两者均于1996年7月3日提交的容许的美国申请序列号08/675,556和08/675,566(重组腺病毒);Grunhaus等人,1992,"Adenovirus as cloning vectors,"Seminars in Virology(第3卷)第237-52页,1993;Ballay等人EMBO Journal,第4页,第3861-65页,Graham,Tibtech 8,85-87,April,1990;Prevec等人,J.Gen Virol.70,42434;PCT WO 91/11525;Felgner等人(1994),J.Biol.Chem.269,2550-2561,Science,259:1745-49,1993;and McClements等人,"Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B,alone or in combination,induces protective immunity in animal models ofherpes simplex virus-2disease",PNAS USA 93:11414-11420,October 1996;以及美国专利号5,591,639、5,589,466和5,580,859,以及WO 90/11092、WO93/19183、WO94/21797、WO95/11307、WO95/20660;Tang等人,Nature,以及Furth等人,Analytical Biochemistry,relating to DNA expression vectors,inter alia.See also WO 98/33510;Ju等人,Diabetologia,41:736-739,1998(慢病毒表达系统);Sanford等人,美国专利号4,945,050;Fischbach等人(Intracel);WO 90/01543;Robinson等人,Seminars in Immunology第9卷,第271-283页(1997)(DNA载体系统);Szoka等人,美国专利号4,394,448(将DNA***活细胞内的方法);McCormick等人,美国专利号5,677,178(使用致细胞病变病毒);以及美国专利号5,928,913(用于基因递送的载体);以及本文引用的其它文件。

优选的病毒载体包括杆状病毒,例如BaculoGold(BD Biosciences Pharmingen,San Diego,Calif.),特别地条件是生产细胞是昆虫细胞。尽管杆状病毒表达系统是优选的,但本领域技术人员应理解,其它表达系统对于本发明的目的起作用,即将HG表达到细胞培养物的上清液内。此类其它表达系统可能需要使用信号序列以便使H5表达到培养基内。

有效剂量:

本文所述的化合物可以以治疗有效剂量施用于对象,以预防PDCoV和/或PEDV相关疾病。剂量取决于接受疫苗的宿主以及因素如宿主的大小、重量和年龄。

用于制剂中的本发明的免疫原性组合物的精确量将取决于施用途径和对象的性质(例如,年龄、大小、疾病的阶段/水平),并且应该根据从业者的判断和根据标准临床技术的每个对象的情况来决定。有效免疫量是足以治疗或预防对象中的PDCoV和/或PEDV传染病的量。

组合物的免疫原性可以通过经由使用本领域已知的任何免疫测定监测用组合物免疫后测试对象的免疫应答来确定。体液(抗体)应答和/或细胞介导的免疫的生成可以理解为免疫应答的指示。测试对象可以包括动物,例如猪、小鼠、仓鼠、犬、猫、兔、牛、马、绵羊、家禽(例如鸡、鸭、鹅和火鸡)和人。

可以通过各种方法分析测试对象的免疫应答,所述方法例如:如通过已知技术例如酶联免疫吸附测定(ELISA)、免疫印迹、免疫沉淀、病毒中和等测定的,所得的免疫血清对免疫原性缀合物的反应性;或者,如通过本领域已知用于测定传染病因子的水平,例如病毒水平(例如,通过培养来自对象的样品)的任何方法,或本领域已知的其它技术确定的,通过保护免疫宿主免于被病原体感染和/或免疫宿主中由于被病原体感染的症状的减弱。还可以通过测量免疫球蛋白针对其的抗原水平来确定传染病因子的水平。传染病因子水平中的降低或传染病症状的改善指示该组合物是有效的。

在体内用于动物之前,可以在体外测试本发明的治疗试剂的所需治疗或预防活性。例如,可以用于确定是否指示特定治疗试剂的施用的体外测定包括体外细胞培养测定,其中来自细胞系的适当细胞或者从患有特定疾病或病症的对象培养的细胞暴露于或以其它方式施用治疗试剂,并且观察治疗试剂对细胞的作用。

可替代地,治疗剂可以通过以下来测定:使治疗剂与易受通过传染病因子的感染但未感染有传染病因子的细胞(从对象或培养的细胞系培养)接触,使细胞暴露于传染病因子,然后确定与治疗剂接触的细胞的感染率是否低于未与治疗剂接触的细胞的感染率。用传染病因子感染细胞可以通过本领域已知的任何方法测定。

另外,可以通过在治疗之前、治疗期间或治疗之后,以合适的时间间隔测量抗体在动物模型或人对象中针对的分子水平来评估治疗剂。可以鉴定分子的量中的任何变化的变化或不存在,并且将其与治疗对于对象的作用相关联。分子的水平可以通过本领域已知的任何方法确定。

在使用本发明的方法和组合物用PDCoV和/或PEDV给动物接种疫苗后,本领域已知的任何结合测定都可以用于评价所得到的抗体和特定分子之间的结合。还可以进行这些测定,以选择对于特定抗原显示出更高亲和力或特异性的抗体。

对受试者的施用:

优选的施用途径包括但不限于鼻内、经口、皮内和肌内。技术人员将认识到,本发明的组合物还可以以一个、两个或更多个剂量,以及通过其它施用途径施用。例如,此类其它途径包括皮下、皮内、静脉内、血管内、动脉内、腹膜内、鞘内、气管内、皮内、心内、叶内、髓内、肺内和***内。取决于所需的治疗持续时间和有效性,本发明的组合物可以一次或多次施用,也可以间歇施用,例如在每天的基础上共几天、几周或几个月,并且以不同的剂量。

包括下述实施例以证实本发明的优选实施方案。本领域技术人员应该了解,下述实施例中公开的技术代表由本发明人发现的在本发明的实践中很好地起作用的技术,并且因此可以被视为构成用于其实践的优选模式。然而,根据本公开内容,本领域技术人员应当了解,可以在所公开的具体实施方案中做出许多改变并且仍然获得相同或相似的结果,而不背离本发明的精神和范围。

实施例

实施例1:PEDV毒株的分离和生产

为了产生猪流行性腹泻病毒疫苗,杀死病毒,首先产生PEDV(分离株)的母种毒培养物。从该母种毒生长PEDV培养物,然后灭活。然后将灭活的病毒培养物与佐剂混合,以便产生猪流行性腹泻病毒疫苗。下述方法用于生产猪流行性腹泻病毒疫苗。

在2013年获得显示出极度腹泻的动物来自动物的或组织。从这些动物生成来自粘膜刮屑的匀浆物,通过0.2微米注射器式滤器过滤,并且将滤液用于接种非洲绿猴肾细胞(VERO)。病毒在PEDV维持培养基的存在下生长,所述PEDV维持培养基含有改良MEM、猪胰蛋白酶、胰蛋白胨磷酸盐肉汤、酵母提取物和HEPES缓冲液。通过检查特征性合胞体形成和细胞单层的融合来评估病毒生长且使其可视化。使用基于Illumina的测序仪技术,对CPE阳性材料进行测序。

为了产生PEDV母种毒培养物(“PEDV MSV”),在BI VERO细胞中分离猪流行性腹泻病毒株(分离株)(PEDV分离株),并且在BI VERO细胞中总共传代19次,然后病毒在2013 EUVERO细胞中生长至第30代。病毒的第30代被稀释并且记录为母种毒,命名为PEDV KV-1251-125-10-OK。

从母种毒中,通过用在PEDV维持培养基中的PEDV KV-1251-125-10-OK MSV感染2013 EU VERO细胞产生PEDV培养物(KV-1251-125-10-OK,在本文中称为“125-10”),所述PEDV维持培养基含有改良的最低必需培养基、猪胰蛋白酶(10μg/ml)、胰蛋白胨磷酸盐肉汤(0.3%)、酵母提取物(0.02%)和1M HEPES缓冲液(2.5%)。2013EU VERO细胞通常以104TCID50/850cm2滚瓶的最小剂量用PEDV(125-10)MSV进行感染。此类培养物可以在无菌一次性滚瓶或微载体珠上生长。将培养物在36℃.±2℃.下温育24至48小时,直至观察到致细胞病变效应(“CPE”)。通常,在感染后12小时内可以看到特征性合胞体,合胞体扩增,细胞单层融合24-48小时,随后为细胞脱落。在温育期间,监测培养物的PEDV诱导的CPE,以确保纯PEDV毒株。如果观察到非典型CPE或者存在任何宏观或微观污染证据,则丢弃培养物。将纯病毒培养物无菌收获到无菌聚丙烯大玻璃瓶(carboy)内。将病毒冷冻解冻以释放细胞结合的病毒粒子,并且通过离心或通过经由0.45微米随后为0.2微米的滤器过滤得到澄清。测试大量病毒收获流体,以确保在灭活之前支原体的不存在。将未立即灭活的收获流体贮存于-70℃或更低温度下。

确定收获的流体的体积并且使流体的温度达到36±2℃。将0.4M的2-溴乙撑胺(BEA)溶液与0.3N NaOH的储备溶液混合,以生成二乙烯亚胺(BEI)储备溶液,然后将其加入收获的流体中,以得到最终浓度为5mM的BEI。将流体连续搅拌最少24小时。加入1.0M硫代硫酸钠溶液,以得到5mM的最终最低浓度,以中和任何残留的BEI。灭活的流体可以贮存于-70±3℃下用于长期贮存,或贮存于4±3℃下用于短期贮存。

用BEI处理后,测试培养物诱导PEDV典型的CPE的能力,以确保病毒的灭活。该任务通过将BEI处理的病毒流体经过Vero细胞,并且检查Vero细胞的任何病毒感染来完成。BEI处理的培养液通常贮存于-70℃或更低温度下,直至灭活测定已完成。

通过将灭活的PEDV培养物与佐剂以20%的包含率彻底共混以形成大量系列,将灭活的病毒配制为佐剂化的疫苗。将大量系列维持在2-8℃下,直至转移到含有一个或十个剂量(每剂量2.0ml)的小瓶内。

实施例2:猪丁型冠状病毒毒株的分离和生产

猪丁型冠状病毒(PDCoV)购自National Veterinary Service laboratory(NVSL),Ames,IA。该病毒在NVSL从2014年来自Illinois的猪肠样品中分离。在5μg/mL TPCK胰蛋白酶的存在下,在无血清培养基中的猪睾丸(ST)细胞上分离病毒。原始病毒在NVSL进行两轮噬斑纯化,并且病毒的第十次传代用作原材料。

将第10代病毒接种并且适应在BI处的猪睾丸(ST)细胞,使用含有具有HEPES的改良的最低必需培养基、胰蛋白磷酸盐胨肉汤、酵母提取物和猪胰蛋白酶的PDCoV维持培养基。将细胞在37℃与5%CO2下温育24-48小时,并且观察PDCoV诱导的致细胞病变效应的证据。当CPE完成并且细胞已脱落时,将烧瓶冷冻解冻一次并且收获裂解产物。然后将收获的材料接种到新的单层上用于继续传代。传代以此类方式继续通过第12次。接种从第12次收获的病毒,并且使其适应命名为“AI-ST 2015”细胞的替代衍生猪睾丸(ST)细胞谱系。以与第11和12次相同的方式,在AI-ST 2015细胞中进行另外四次病毒传代,以获得P16病毒。将P16病毒接种到滚瓶内,得到最终的“P17”母种毒分离株,将其收获,通过0.2μm滤器过滤,合并以获得大量病毒原种并且贮存于-60℃以下。母种毒“P17”的平均滴度被确定为4.91log10FAID50/mL。

实施例3:PEDV分离株和PDCoV分离株的基因组序列分析

样品制备和分析:

在提取之前,用DNA酶和RNA酶的混合物预处理病毒组织培养上清液,以去除残留的宿主细胞基因组核酸。然后使用病毒RNA提取试剂盒(Qiagen,目录#52906),从核酸酶处理的样品中提取病毒基因组RNA。在提取后,再次用DNA酶处理样品以进一步富集病毒基因组RNA。随后,通过随机引发的逆转录和Klenow片段处理,将病毒基因组RNA转换为双链cDNA(ds cDNA)。然后使用 XT文库制备试剂盒(目录#FC-131-1024),将ds cDNA产物用于生成基于Illumina 测序仪的测序的文库。每个样品在5'和3'末端两者上均用独特标签加上条形码,以使生物信息错误框并的机会降到最低。该文库使用500循环试剂盒(目录#MS-102-2003)在测序仪上运行,并且使用NextGene(版本2.3.4)和Sequencher软件(版本5.1)的组合分析数据。选择高质量序列作为含有中值Q得分大于25的那些,并且在3'末端处以不超过3个未调用碱基或测量小于16的Q得分的3个连续碱基的切断进行修剪。然后使用在35bp段上85%或更高匹配的标准从头组装序列,以生成关于每种分离株的假定的完全基因组。然后通过基于模板的比对验证关于各自的假定的完全基因组序列,以验证单核苷酸多态性(SNP)或可变的小***/缺失。

对于PEDV分离株1251-125-10,在修剪低质量数据后生成平均长度为136bp的总共570,253个序列。在那些序列中;484,247(84.9%)组装成长27,995bp的单个重叠群,其通过BLASTn分析揭示与单链RNA甲型冠状病毒PEDV的强烈同一性。总共11个位置显示出在单个核苷酸或小***/缺失处的多态性,这些位置在表1中列出。

表1:分离株1251-125-10“125-10”中的多态性残基

将1251-125-10(SEQ ID NO:29)的假定的完全/接近完全的PEDV基因组与PEDV的最接近的中国AH2012(GenBank登录号:KC210145)和北美Colorado 2013分离株(GenBank登录号:AGO58924)进行比对。与两种毒株的同一性超过99.2%,指示与两者均在基因群2a中的两种毒株非常密切的关系。

接下来,检查免疫原性刺突蛋白序列与PEDV刺突蛋白的较大GenBank储库的蛋白质同一性/相似性。再次,提交的最接近的GenBank分离株衍生自由University ofMinnesota Veterinary诊断实验室保藏的北美Colorado 2013毒株(GenBank登录号:AGO58924),其显示出超过99.5%的同一性(1380/1386个相同的氨基酸)(SEQ ID NO:X)。在6个氨基酸变化中,1个是由于在位置23,101处的多态性,其在位置838处大多数编码CGA(Arg)且小部分编码CAA(Gln)。北美Colorado 2013年毒株在该位置处含有Gln。

对于PDCoV,对12个个别分离的样品进行测序。在病毒颗粒保护的核酸消化(RNA酶+DNA酶)后提取材料,以便富集病毒RNA。在提取后,用DNA酶处理核酸以富集RNA。通过使用随机六聚体引发的逆转录和klenow处理生成双链cDNA。然后将这些产物用于文库生成。

使用 XT文库制备试剂盒(Illumina,目录#FC-131-1024),通过文库生成处理样品用于基于测序仪的测序。每个样品在5'和3'末端两者上均用独特标签加上条形码,以使生物信息错误框并的机会降到最低。该文库使用500循环试剂盒(Illumina,目录#MS-102-2003)在测序仪上运行,并且使用测序软件(SoftGenetics,LLC,版本2.3.4.2)和软件(Gene Codes Corp.,版本5.1)的组合分析数据。选择高质量序列作为含有中值Q得分大于25的那些,并且在3'末端处以不超过3个未调用碱基或测量小于16的Q得分的3个连续碱基的切断进行修剪。然后使用在35bp段上85%或更高匹配的标准从头组装这些序列,并且与参考文件比对,使用85%或更高的标准匹配35bp的伸展。用于比对的参考序列来自从头序列组装或关于Carthage和NVSL PDCoV毒株(NSVL,猪丁型冠状病毒毒株USA/IL/2014/026PDV_P11,Genbank号KP981395.1)的测序仪衍生的参考。

总的来说,每种BI病毒分离株和其它毒株(“Carthage”原始分离株和NVSL毒株)之间的遗传相似性很高(>99.8%同一性),并且NVSL和BI-ST细胞生长的病毒变体之间的差异显示出来甚至更少的变化(>99.95%的同一性)(表2)。还参见分别示意性地显示了关于PDCoV分离株/毒株的完全基因组核苷酸邻近树、以及关于PDCoV分离株/毒株的刺突氨基酸邻近树的图1和2。比例尺指示p距离。

NVSL和BI-ST细胞生长的病毒的变体之间的相似性很可能是由于一些样品的共享起源。一个值得注意的序列差异是在培养的PDCoV_2201-1分离株内刺突(氨基酸位置229-231)中的框内9bp缺失的存在;但在原始匀浆物或来自猪的材料中未观察到。这个位置看来是IN/DEL变异性的“热点”,因为与“Carthage”分离株没有已知连接的BI PDCoV-5.0327分离株,在“BI-ST”细胞系中也显示出相同的缺失,但在NVSL-ST细胞系中则没有。这些变化可能指示病毒的组织培养适应性。

表2:每种BI病毒分离株之间的遗传相似性。

实施例4:整个PEDV和PDCoV分离株的灭活

通过在VERO细胞中传代来测试每批PEDV病毒或库的灭活。75cm2的24小时细胞培养物用2.0mL灭活的PEDV流体接种,并且在36±3℃下维持48小时。一个VERO细胞烧瓶保持未接种。对于阳性病毒对照,用阳性对照PEDV接种VERO细胞的一个培养物。在温育期结束时,检查细胞单层的PEDV典型的CPE。将材料冷冻和解冻三次,然后将2ml每种材料接种到一天大的VERO细胞上。培养物应该在37±2℃下维持48小时。在第二次传代之后,进行第三次传代。在温育和传代后,如通过免疫荧光染色的缺乏确定的,在BEI处理的病毒液中不存在病毒感染的细胞构成令人满意的灭活测试。用阳性对照病毒接种的对照细胞应该显示PEDV典型的CPE,并且未接种的烧瓶不应显示PEDV CPE的证据。

PDCoV病毒收获物在37℃下用5mM BEI灭活24小时。灭活后,用硫代硫酸钠中和收获物。通过灭活收获物的TCID50评估来确认灭活。在灭活材料中未检测到活病毒。使用10kd中空纤维PES药液筒(GC Healthcare目录#UFP-10-C-3MA)浓缩灭活的中和的病毒收获物。用改良的最低必需培养基无菌稀释浓缩的抗原(最终浓度=10X)。将一部分稀释的抗原与EMULSIGEN (MVP Laboratories,项目#10005408)组合,以获得12.5%的制剂。

实施例5:

编码且表达PDCoV刺突抗原的重组杆状病毒的构建

PDCoV S1-IgG2a Fc杆状病毒的制备

使用引物P2967167A(SEQ ID NO:13)和P2967167B(SEQ ID NO:14),通过PCR从质粒DNA扩增猪丁型冠状病毒(PDCoV)刺突(aa 1-673)的S1区域。使用引物P2967167C(SEQ IDNO:15)和P2968014E(SEQ ID NO:16),从质粒DNA扩增包含短GGS接头和铰链区的猪IgG2a的Fc区。使用引物P2967167A(SEQ ID NO:13)和P2968014E(SEQ ID NO:16),通过重叠-延伸PCR融合两个片段,以生成含有起始密码子紧5'的Kozak共有序列的PDCoV S1-IgG2a Fc编码序列(SEQ ID NO:17)。参见图3.A中的示意图。最终的编码序列侧面为BamHI和NotI限制性位点,以促进克隆到杆状病毒转移质粒pVL1393内。一旦完成,将质粒pVL1393-PDCoV S1-IgG2a Fc与线性化的BaculoGold杆状病毒DNA一起用于转染Sf9昆虫细胞,以产生重组杆状病毒。

PDCoVS BD Baculodisplay杆状病毒的制备

将来自猪丁型冠状病毒(PDCoV)的PDCoV刺突基因克隆到来自质粒DNA的两个重叠片段(N末端和C末端)内。使用引物P2967002C(SEQ ID NO:23)和P2967002D(SEQ ID NO:24)扩增N末端片段,而使用引物P2967002E(SEQ ID NO:25)和P2967002F(SEQ ID NO:26)扩增C末端片段。扩增N末端和C末端片段,以去除天然信号序列和C末端尾部。使用引物P290194A(SEQ ID NO:19)和P2967002D(SEQ ID NO:20),通过重叠-延伸PCR(OE-PCR),将N末端片段与苜蓿银纹夜蛾核多角体病毒(AcNPV)gp64信号序列(O2967002A)(SEQ ID NO:21)融合。使用引物P290194B(SEQ ID NO:20)和P2967002E(SEQ ID NO:25),通过OE-PCR将C末端片段与AcNPV gp64C末端尾部编码序列(O2967002B)(SEQ ID NO:22)融合。使用引物P290194A(SEQID NO:19)和P290194B(SEQ ID NO:20),通过OE-PCR融合两个所得到的片段,以生成含有起始密码子紧5'的Kozak共有序列的PDCoV刺突BaculoDisplay(PDCoVS BD)编码序列(SEQ IDNO:27)。参见图3B中的示意图。最终的编码序列侧面为BamHI和NotI限制性位点,以促进克隆到杆状病毒转移质粒pVL1393内。一旦完成,将质粒pVL1393-PDCoVS BD与线性化的BaculoGold杆状病毒DNA一起用于转染Sf9昆虫细胞,以产生重组杆状病毒。

实施例6:

包含PEDV刺突抗原的药物组合物(疫苗)的制备:

对于灭活的PEDV材料,PEDV病毒收获物使用5mM BEI灭活最少24小时,澄清且0.45μm过滤。

中和后,加入各种佐剂,并且生成下述疫苗/药物组合物。

实施例7:

包含PDCoV刺突抗原的药物组合物(疫苗)的制备:

PDCoV抗原(BacluloS-BD-PDCoV和BaculoS-FC-PDCoV)的分批在3L旋转器中生长。对于感染,SF+细胞以大约1.0-2.1的MOI用病毒进行接种。将烧瓶在27℃下温育,伴随设定在100rpm下的搅动。在感染后五天起始收获。在收获时,细胞活力在24%和26%之间,并且活细胞/mL在0.31 x 106和0.36 x 106之间。通过以10,000x g离心10分钟来澄清收获流体且进行0.2μm过滤。澄清的收获流体与D组合,以获得12.5%的制剂。

实施例8:

用灭活的PDCoV和杆状病毒刺突疫苗接种猪和血清学应答的评价

研究目的是评估PDCoV疫苗原型在常规仔猪中的血清学应答。已生成了几种原型疫苗,其中PDCoV的刺突蛋白在各种主链中表达。另外,已生成了全细胞灭活的病毒制剂。

关于获得实验组的解释参见下表3。第1-4组随机化并且保存在同一房间中。另外,在当前研究中包括在大约4周龄时断奶的严格对照动物(第5组)。在D0时,将猪随机分成四组,并且施用2mL剂量的每种原型PDCoV疫苗或安慰剂。在D21时,猪接受每种原型疫苗的第二次或加强施用。在每次疫苗接种的处理施用之前和在D35时,从猪中收集血清和经口流体。测定样品的血清转换证据。每天通过房间记录一般健康观察。在疫苗施用之后,观察注射部位的反应最少三天。

表3:实验组

N(猪) 疫苗
1 10 BaculoS-BD-PDCoV
2 11 全细胞PDCoV
3 11 安慰剂
4 10 BaculoS-FC-PDCoV

如通过IFA检测的疫苗接种后的血清学应答

间接免疫荧光测定(IFA)

在1x磷酸盐缓冲盐水(PBS;Gibco c#10010-023)中制备每个样品从1:40到1:320的两倍连续稀释物。将100μl每种稀释的样品加入PDCoV感染的板中,并且在37℃下温育1小时。温育后,去除血清,且用1xPBS洗涤单层细胞两次。然后使用10μl/孔的1:100稀释的FITC-缀合的-山羊抗猪IgG抗体将细胞染色。与未感染的对照孔相比,滴度被报告为显示PDCoV染色的最大血清稀释度。

在D-8、D21和D35时的血清学应答在下表4中按组呈现。表5中呈现了按日和组的最小二乘平均值和检测的频率。在疫苗接种之前,没有动物具有可检测的血清学应答。(也参见图4)。

在信号疫苗接种后,20-73%的动物对用BaculoS-BD原型接种的动物具有应答。到D35时,用全细胞或BaculoS-FC原型接种疫苗的所有动物都具有可检测的应答。用BaculoS-BD原型接种疫苗的动物中仅50%具有到D35时可检测的应答。

在用原型或安慰剂接种的猪的血清学应答之间没有观察到在D-8时的显著差异。到D21和35时,血清学应答在接受原型疫苗的动物中更高(对于所有组在所有天时p<0.002)。在数字上,与BaculoSBD构建体相比,BaculoS-FC和全细胞原型疫苗看来到D35时刺激更高的IgG应答。

表4:如通过IFA测量的按天和组的血清学数据

*指示该值与安慰剂组明显不同(Dunnett方法)

如通过基于S1-IgG2a-Fc的ELISA检测的疫苗接种后的血清学应答

PDCoV IgG ELISA

将纯化的PDCoV-S1-IgG2aFc抗原(BIVI L#3091-141;0.2mg/mL)在碳酸盐缓冲液(BIVI L#3144-180)中1:6666.67稀释。ELISA板(高结合96孔板;Greiner目录#655061)用100μl/孔稀释的抗原包被,并且在4℃下温育过夜。使用自动洗板机,用300μl/孔的TBST(0.05%吐温20)将板洗涤五次。在洗涤步骤后,每孔加入150μl阻断溶液(酪蛋白阻断剂,Thermo目录#37532),并且使板在15-30℃下温育1小时。在酪蛋白阻断剂中以1:200的稀释度制备初始血清稀释物(阴性对照和测试样品)。温育后,从板中去除酪蛋白阻断剂,并且从1:200到12800制备测试样品的连续两倍稀释物(在酪蛋白阻断剂中制备)。阴性对照的连续两倍稀释物由1:200直到1:204800制备。在板上制备稀释物,伴随100μl/孔的最终体积。将板在37℃下伴随振荡(100rpm)温育1小时。温育后,如先前所述洗涤板。制备HRP缀合的山羊抗猪IgG(H+L)(Jackson ImmunoResearch目录#114-035-003)的1:2000稀释物(在酪蛋白阻断剂中),并且向每个孔中加入100μl。使板在37℃下伴随以100rpm的振荡温育1小时,然后如先前所述洗涤。为了检测,每孔加入100μl/孔的制备的TMB底物(KPL目录#50-76-00)。将板在15-30℃下温育10分钟。用100μl/孔的1N HCl终止反应,并且立即用自动读板仪在450nm处读数。

通过下述计算生成结果。首先,计算来自最终五个阴性血清稀释孔的平均和标准差光密度(OD)读数。然后将截断确定为平均值加上三个标准差。将每个样品光密度读数(在其初始稀释度下)除以该板的本底截断。数据报告为信号:本底比。

在D-8、D21和D35时的血清学应答按组呈现。表5中呈现了按日和组的最小二乘平均值和检测的频率(也参见图5)。在疫苗接种之前,没有动物具有可检测的血清学应答。

第二次疫苗接种后,40-45%的动物都具有应答,与组无关。到D35时,用全细胞或BaculoS-FC原型接种疫苗的所有动物都具有可检测的应答。在用原型或安慰剂接种的猪的血清学应答之间没有观察到在D8或D21时的显著差异。到D35时,血清学应答在接受原型疫苗的动物中更高(对于第1、2和4组,分别为p=0.0039、<0.0001或<0.0001)。与IFA结果相似,与BaculoS-BD构建体相比,BaculoS-FC和全细胞原型疫苗到D35时刺激更高的IgG应答。

表5:如通过IgG ELISA测量的按天和组的血清学数据

*指示该值与安慰剂组明显不同(Dunnett方法)

实施例9:

PEDV杆状病毒疫苗的功效

下述研究评估了对用两个2-mL剂量的杀死的猪流行性腹泻病毒(PEDV)疫苗或杆状病毒构建体疫苗的疫苗接种的血清学应答,如在给以三周龄的猪施用任一疫苗后测量的。主要结果是对于在处理的猪中的疫苗接种后收集的血清样品通过荧光聚焦中和(FFN)测试的血清学。

研究组包括:T01=PBS(n=10);T02=6.93 log TCID50/ml BEI PEDV+20%(n=20);T03=具有PEDV刺突Ag的杆状病毒(n=9);具有PEDV刺突Ag的6x浓缩杆状病毒(n=10);具有PEDV刺突Ag的胰蛋白酶杆状病毒(n=10);以及有条件批准的杀死的阳性对照疫苗(POS CON)(n=10)。在D0时,猪在右颈部中肌内施用2-mL治疗。第二次治疗对于T01-T05在左颈部中在D14时施用,并且对于T06在D21时施用。

PEDV2a-BD的制备

在该研究中,将来自猪流行性腹泻病毒(PEDV)2a的刺突基因克隆到来自质粒DNA的两个重叠片段(N末端和C末端)内。使用引物P1360110A(SEQ ID NO:37和PEDV-S2-R(SEQID NO:38)扩增N末端片段,而使用引物PEDV-S1-F(SEQ ID NO:39)和P1360110B(SEQ IDNO:40)扩增C末端片段。扩增N末端和C末端片段,以去除天然信号序列和C末端尾部。使用引物P290194A(SEQ ID NO:45)和PEDV-S2-R(SEQ ID NO:38),通过重叠-延伸PCR(OE-PCR),将N末端片段与苜蓿银纹夜蛾核多角体病毒(AcNPV)gp64信号序列(O1360110C)(SEQ ID NO:41)融合。使用引物P290194B(SEQ ID NO:43)和PEDV-S1-F(SEQ ID NO:44),通过OE-PCR将C末端片段与AcNPV gp64C末端尾部编码序列(O1360110D)(SEQ ID NO:42)融合。使用引物P290194A(SEQ ID NO:45)和P290194B(SEQ ID NO:43),通过OE-PCR融合两个所得到的片段,以生成含有起始密码子紧5'的Kozak共有序列的PEDV刺突BaculoDisplay(PEDVS BD)(SEQ ID NO:46)。最终的编码序列侧面为BamHI和NotI限制性位点,以促进克隆到杆状病毒转移质粒pVL1393内。一旦完成,将质粒pVL1393-PEDVS BD与线性化的BaculoGold杆状病毒DNA一起用于转染Sf9昆虫细胞,以产生重组杆状病毒。参见图6A.PEDV 2a BDBaculodisplay杆状病毒的示意图。

表6:PEDV原型制剂和对照

BEI=二乙烯亚胺

*PEDV刺突信号序列和C末端尾部替换为杆状病毒gp64等价物。在昆虫细胞中产生重组PEDv刺突-展示杆状病毒。收获受感染的培养物并且通过离心和0.2-μm过滤澄清。将澄清的收获材料用5mM BEI在37℃下灭活72小时,然后通过离心和0.2-μm过滤澄清。

血清学:

疫苗接种后的血清转换(D28和D35)在20%用由20% BCL佐剂化的PEDv疫苗接种疫苗的猪(T02;表7)和60%用胰蛋白酶生长的PEDV SPIKE-杆状病毒接种疫苗的猪(T05;表7)中发生。对于所有处理组血清阳性的猪≥1:20的几何平均滴度呈现于下表7中。按所有猪的处理组的滴度频率分布呈现于表8中。

表7:对于血清学应答的猪,按组的血清阳性猪的比例和几何平均滴度

具有≥1:20应答的猪 几何平均滴度
T01 0/10(0%) 不可用
T02 4/20(20%) 1:30.7
T03 0/9(0%) 不可用
T04 0/10(0%) 不可用
T05 6/10(60%) 1:35.5
T06 7/10(70%) 1:41.9

表8:按组的滴度频率分布

结论:

在实验疫苗的两次施用后,在20%的T02猪中发生血清转换,所述实验疫苗用由BEI灭活且由20% BCL佐剂化的6.93log10TCID50/mL PEDV配制。在用PEDV刺突糖蛋白配制的实验性重组胰蛋白酶生长的杆状病毒疫苗的两次施用后,在60%的T05猪中发生血清转换。

实施例10:

PEDV 2a BD杆状病毒疫苗的功效

该研究的主要目的是评估原型PEDV杆状病毒疫苗在常规母猪模型中的功效。将36头母猪随机分成5组。母猪在分娩前5周和2周时用实验性PEDV疫苗、安慰剂、商购可得的阳性对照进行肌内疫苗接种,或未接种疫苗(严格对照)。所使用的组和疫苗的简要描述在下表9中列出。在妊娠期、分娩期和攻击期自始至终,从母猪和猪中收集血清和粪便样品,以监测抗PEDV抗体和PEDV RNA的存在。每天记录一次临床体征。

该研究的主要结果参数是猪死亡率。基于69%的猪死亡率、临床体征的存在以及来自安慰剂组中的猪的粪便材料中PEDV RNA的鉴定,用于本研究的攻击模型被视为有效的。在母猪中使用两个剂量的BaculoS-NT构建体能够显著减少猪的死亡率。相比之下,目前市场上的PEDV疫苗也能够减少猪的死亡率;然而,减少并不显著。由于BaculoS-NT原型不在胰蛋白酶的存在下生长,并且用12.5%D佐剂化,因此预期该原型将是用于生产的优选形式并且具有21天的撤药期。

该研究的次要结果参数是疫苗原型预防猪中的临床体征的能力。由于所有猪中的大于93%对于最少连续两天具有纯液体腹泻,因此疫苗原型或商购可得的产品无一能够预防严重临床体征的发作。类似地,疫苗无一能够减少猪中的临床体征的持续时间。

表9.研究设计:

母猪在分娩前5周和2周时用实验性PEDV疫苗、安慰剂、商购可得的阳性对照进行肌内疫苗接种,或未接种疫苗(严格对照)。在妊娠期间,在每次疫苗接种前收集血液和粪便样品,且分别测定血清转换和脱落的证据。每天对每头母猪记录临床观察。

猪死亡率:

用强毒PEDV分离株攻击后的猪死亡率是用于评价疫苗功效的主要结果参数。在攻击期间按组的死亡率概括呈现于下表10中。按组和天数的生存性呈现于图7中。'按组和天数的存活猪的百分比')。在D0时,使用适当大小的无菌针头和注射器,将2mL剂量的疫苗施用于健康母猪的右颈部的肌肉组织内。在D21时,该过程是相同的,除了在颈部的左侧上给予注射之外。

攻击材料是强毒PEDV原种材料(PEDV 1251-140-4;p5;L#2842-174;滴度=2.02 x105TCID50/mL),其在PEDV病毒生长培养基中稀释至1.0 x 103的滴度。(具有HEPES、胰蛋白胨磷酸盐肉汤和酵母提取物的改良的最低必需培养基)。与之前的研究相似,大多数死亡率在暴露于攻击材料的8天内可见。

总之,所有组在感染后48小时内具有存在于大多数猪中的临床体征的相似趋势。临床体征在所有组中超过50%的猪存在直到DPC6。从DPC7到DPC11,临床体征得到消除。到DPC13时,在任何猪中都没有观察到临床体征。

分析:

生物统计学。所有数据都输入版本9.4内用于分析。生成按处理组的数据列表和概括统计学,包括频率分布。适当时进行掺入窝和居住效应的分析。列出了按组和窝的攻击后的猪死亡率,并且描述统计学平均值、中值、最小值和最大值用于按组的概括。使用具有皮尔森卡方作为过分散的广义线性模型将按组和窝的死亡率分析。然后,估计与安慰剂组相比,关于每个处理组的基于模型的预防分数和95%置信区间。具有皮尔森卡方作为过分散的广义线性模型与死亡率数据再一次拟合,加上年龄、处理得分和奇偶性(parity)作为固定效应,以调查这些因素是否对死亡率有任何作用。列出了研究随机化数据。列出了关于猪的粪便得分,并且使用频率分布按组和天数进行概括。概括在攻击之前和攻击期间分别完成。关于母猪的临床体征(粪便、呕吐物、注射部位病变、无乳和处理的)得分使用频率分布按组和天数分别在分娩前和分娩后列出且概括。通过窝、组和天数使用描述统计学中值、最小值和最大值,列出且概括猪重量数据。平均日增重(ADWG)通过混合模型进行分析,其中组和分娩时的重量作为固定效应;窝和用于居住的房间作为随机效应。估计最小二乘平均值及其在组间的对比;估计相应的标准误和95%置信区间。使用按通过死亡率分开的组的每个变量的计数列出且概括关于猪的尸检数据,包括器官的病理学数据以及盲肠的内容物、结肠、小肠和肺部病变,以及胃记录。是否存在任何病变也使用计数和按组的百分比进行概括。首先列出实验室数据,包括关于母猪的PED病毒血清抗体滴度(FFN)、分别关于猪和母猪的血清IgA全细胞ELISA、关于母猪的IgA和IgG baculos ELISA、关于母猪和猪的IgA和IgG IDEXX ELISA和PCR。接下来,使用几何平均值(FFN)(尽管对于FFN,未提供标准差)或平均值(ELISA、PCR)计算描述按组和天数的统计学平均值、最小值、最大值和标准差。接下来,使用具有窝和用于居住的房间作为随机效应的混合模型,来估计按组和天数的最小二乘平均值、标准误和95%置信区间。估计组对之间的最小二乘平均值的对比和相应的95%置信区间。

表10:按组的猪死亡率

该研究的主要结果参数是猪死亡率。基于该参数,在母猪中使用两个剂量的BaculoS-NT构建体能够显著减少猪的死亡率(PF=0.48)。相比之下,前市场上的PEDV疫苗也能够减少猪的死亡率;然而,减少并不显著(第4组)。由于BaculoS-NT原型不在胰蛋白酶的存在下生长,并且用12.5%D佐剂化,因此预期该原型将是用于生产的优选形式并且具有21天的撤药期。

该研究的次要结果参数是疫苗原型预防猪和母猪中的临床体征的能力。由于所有猪中的大于93%对于最少连续两天具有纯液体腹泻,因此疫苗原型或商购可得的产品无一能够预防严重临床体征的发作。类似地,疫苗无一能够减少猪中的临床体征的持续时间。在临床体征消除后,在DPC14时12-33%的动物中鉴定出PEDV RNA。这提示疫苗接种无法保护不受定殖。在1-3头母猪/组中,至少连续两天观察到严重腹泻。这指示疫苗不能完全保护母猪不受暴露于由受攻击的猪脱落的大量病毒。另外,大多数母猪(14/16)具有在肠系膜***中存在的可检测量的病毒,指示疫苗接种不能预防定殖。

大多数接种疫苗的母猪在两个剂量的疫苗后具有可检测的IgG(和FFN)应答;与安慰剂疫苗接种的对照相比,滴度更高(数据未显示)。由于IgG ELISA和FFN结果非常相似,很可能FFN测定主要检测IgG。尽管IgG可以中和病毒,但它不太可能是PEDV疫苗功效的唯一决定因素,因为组间的明确差异并不明显。

基于如测量的IgA ELISA(IDEXX)的IgA水平,幼稚和接种疫苗的母猪到DPC13/14时具有相似的血清和乳IgA滴度。然而,在接种疫苗的动物中注意到在DPC3时收集的血清、初乳和乳中增加的IgA滴度。由于来自接种疫苗的母猪的存活猪也具有最高的IgA滴度,该数据支持以前发表的文献,强调IgA在肠道感染中的重要性。(参见图8)。

实施例11:

PEDV2b杆状病毒疫苗的制备

来自猪流行性腹泻病毒(PEDV)2b的刺突基因以两个重叠片段(N末端和C末端)制备。使用引物P290194A(SEQ ID NO:48)和P3183131B(SEQ ID NO:49),从含有苜蓿银纹夜蛾核多角体病毒(AcNPV)gp64信号序列与PEDV 2b刺突的氨基酸22-400的融合物的合成DNA扩增N末端片段。使用引物P3183131A(SEQ ID NO:50)和P290194B(SEQ ID NO:51),从已经含有AcNPV gp64C末端尾部编码序列的pVL1393-PEDVS BD质粒DNA扩增C末端片段。使用引物P290194A(SEQ ID NO:48)和P290194B(SEQ ID NO:51),通过OE-PCR融合两个所得到的片段,以生成含有起始密码子紧5'的Kozak共有序列的PEDV 2b刺突BaculoDisplay(PEDVS 2bBD)编码序列(SEQ ID NO:52)。最终的编码序列侧面为BamHI和NotI限制性位点,以促进克隆到杆状病毒转移质粒pVL1393内。一旦完成,将质粒pVL1393-PEDVS 2b BD与线性化的BaculoGold杆状病毒DNA一起用于转染Sf9昆虫细胞,以产生重组杆状病毒。参见图6B.PEDV2a BD Baculodisplay杆状病毒-蛋白质图的示意图。

实施例12:PEDV和PDCoV功效研究

设计以下研究以评价杀死的PEDV疫苗和/或杀死的PDCoV疫苗或其它原型疫苗在猪中的功效。主要结果参数是用猪流行性腹泻病毒(PEDV)和/或PDCoV攻击后的仔猪死亡率。次要结果参数是母畜(dam)血清学。测量的其它参数包括:疫苗接种后母猪中的临床体征(包括ISL);疫苗接种后母猪中的病毒脱落(经由qRT-PCR);仔猪中的临床体征;以及仔猪中的PEDV和/或PDCoV血清学

PEDV研究:

在分娩前4周和2周(D0和D14)时,通过三种途径(肌内、鼻内和经口)给每只妊娠母畜施用2mL下述处理之一:T01(阴性对照,NC)磷酸盐缓冲液盐水;T02(BEI-VH)用20% BCL佐剂化的;T03(严格对照,SC)充当未接种疫苗/未攻击的对照。每组使用8只动物,排除具有4只动物的T06。在D35或D36时,用1mL 2.0log10TCID50/mL PEDV病毒收获物经口攻击猪。在攻击期过程中每天观察母畜和猪中的临床体征(呕吐和腹泻)。在分娩前四周和两周(D0和D14)时、在仔猪攻击前一天(D34或D35)和脱离测试当日(D57),从母畜中收集血清。

在D0和D14时,将PEDV原型免疫原性组合物施用于母猪。在每次疫苗接种时,母猪接受总共6mL材料,其中2mL通过肌内、鼻内和经口途径施用。对于肌内施用途径,将2mL注射剂给予在耳下的颈部肌肉组织内。用于施用的颈部侧面交替用于初始和加强疫苗接种。对于经口施用途径,使用附接到注射器的8Fr聚丙烯导管(2.7mm直径×254mm长度),在尾部口咽上递送2ml。对于鼻内途径,使用附接到注射器的4.5英寸导管,将1ml注射到每个鼻孔内。

表11:PEDV实验性免疫原性组合物和对照产品

表12:PEDV攻击材料

疫苗功效:

猪死亡率:用强毒PEDV分离株攻击后的猪死亡率是用于评价疫苗功效的主要结果参数。下文列出了在攻击期过程中按组的死亡率概括。在T01(NC)中具有55%的死亡率和所有窝都受影响,该攻击被视为足够强毒的。与T01(NC)相比,T02(BEI-VH)证实猪死亡率中的数目减少,具有0.20(-0.550,0.586)的PF(95%CI)。由于95%CI(-0.550,0.586)包括零,因此减少并非统计学显著的。(参见表13)。

在该研究中,二项式外变化是明显的,当利用潜在的二项式分布时,导致关于T02(BEI-VH)PF的宽置信区间。死亡率在一组内的窝中变化很大,包括对于T02(BEI-VH)从0%到100%。

在尸检时获取肠样品或肠内容物,并且通过qRT-PCR测试以检测PEDV抗原。在峰值死亡率期间从动物测试的样品中,在55.5%的样品中检测到PEDV。

表13:

*基于受影响的T01(NC)比例。

**NC=未计算的。基于研究设计,关于T02(BEI-VH)可能的置信区间

母猪血清学:

荧光聚焦中和(FFN)测定:FFN测定用于评价在疫苗接种和攻击后的母畜病毒中和应答。对于在其下从母猪收集血液的天数,按组列出的几何平均滴度呈现于下文。

在两个剂量的疫苗后,T02(BEI-VH)中的2/8(25%)母猪具有可检测水平的中和抗体。在任何其它组中未观察到可检测水平的中和抗体。

在横向暴露于PEDV后,暴露的处理组中的所有母猪都具有可检测水平的中和抗体。在测试自始至终,T03(SC)组中的动物保持血清反应阴性。在D57时的几何平均滴度(暴露后大约21天)指示,与T01(NC)相比,疫苗接种导致数目更高的滴度。T02(BEI-VH)组中的母猪具有613的GMT,与T01(NC)中母猪200的GMT相比,其是高大约3倍的滴度(p=0.005)。由于T02(BEI-VH)组中的多个样品在测试的最高稀释度(1:640)下具有可检测的中和抗体,这些结果可能代表组间差异的保守估计。

表14:母猪血清学

当所有值都<20时,几何平均滴度呈现为<20。否则,对于GMT计算,<20的值被设为10

**关于T01(NC)和T02(BEI-VH)的D57GMT是反向变换的最小二乘平均值

基于S1的ELISA数据:

基于S1的ELISA用于评价母畜在接种疫苗和攻击后对PEDV刺突蛋白的应答。对于在其下收集样品的天数,关于初乳、乳和血清的测定结果按组列出。

在猪攻击时,与T01(NC)中的母猪相比,T02(BEI-VH)中的母猪在血清中具有显著更高的几何平均滴度(p=0.0005)。在暴露于PEDV后,注意到两组之间更大的显著差异(p<0.0001)。

在T02(BEI-VH)和T04(NC)之间未观察到初乳和乳中的抗PEDV IgA的几何平均滴度中的显著差异。

表15:

*关于T01(NC)和T02(BEI-VH)的GMT是反向变换的最小二乘平均值

猪血清学:

在尸检时从猪中收集血清,以评估中和抗体的存在。下表16呈现了按组的阳性猪的几何平均FFN滴度。表16还包括表示为GMT大于或等于20的猪的数目超过测试动物的数目的检测频率。对于所有可用样品进行测试。由于死亡和尸检之间的时间差异,无法获得来自众多猪的样品。

下文列出了按死亡率状态(死亡:是/否)和组(总体)的FFN滴度的描述统计学。总的来说,不管尸检的时间,接种疫苗的组中相似比例的猪发生血清转换(或具有母体抗体)。然而,在T01(NC)中,与对于研究的持续时间存活的猪(43%)相比,在脱离测试前死亡的更高百分比的猪具有滴度(88%)。

当按组察看猪总体滴度时,死亡率估计值比例与关于T02(BEI-VH)的总体FFN百分比反相关。

表16:

猪(死亡=是) 猪(死亡=否) 总体 死亡率估计值比例
NC 55(28/32;88%) 33(9/21;43%) 63(37/53;59%) 0.55
BEI-VH 44(23/42;55%) 50(9/16;56%) 64(32/58;55%) 0.44

*GMT(滴度≥20的动物数目/测试的总体猪;百分比);注意,并非从所有猪中获得血清。

攻击后的临床观察:

猪粪便得分:在下文列出了关于猪中的异常粪便观察持续时间的描述统计学,按组和死亡率状态(死亡:是/否)。总的来说,具有相同死亡率状态的猪中的异常粪便得分的中值持续时间在组中是相似的。在死亡或实施安乐死的动物中,存在异常粪便得分的数目上较短的中值持续时间。这种趋势在T01(NC)猪中最明显,并且可能继发于大多数这些动物在攻击后第一周内死亡的事实。

表17:

猪中的粪便得分的严重性概括于在下文的频率表18中。在所有处理组中,在攻击后的至少一次观察期间,大部分猪(>91%)呈现粪便得分2。

表18:

结论:

与T01(NC)组相比,在T02(BEI-VH)中观察到猪死亡率中的20%减少。在该研究中尝试了三种施用途径。尽管使用了3种途径,但不预期除IM外的途径促成基于佐剂和疫苗制剂的T02(BEI-VH)的功效。总的来说,具有6.04log TCID50/ml的最小灭活前滴度、用20%佐剂化的灭活PEDV疫苗,看来在仔猪和母猪中诱导更好的免疫应答。优选的疫苗接种方案是对于3周龄或更大的仔猪的IM施用途径,以2周的间隔的3个2ml剂量。在T02(BEI-VH)中未观察到疫苗接种后母猪中的临床体征,并且在其它处理组中受到限制。疫苗接种的使用看来不影响出生的猪存活的百分比(数据未显示)。

通过两种分开的测定(聚焦荧光中和、基于S1的ELISA)评估作为次要参数的母畜血清学。两种测定均指示,与T01(NC)相比,疫苗接种和暴露后T02(BEI-VH)中的滴度的显著增加。由于FFN测定的已知局限性,还通过基于S1的ELISA测试样品。选择该ELISA是因为刺突蛋白的S1结构域预期含有中和表位。

在横向暴露于PEDV后,暴露的处理组中的所有动物都具有可检测水平的中和抗体。与T01(NC)动物相比,T02(BEI-VH)中的母猪具有高大约三倍的滴度。这是疫苗的使用刺激了初始的初级应答并且在暴露于攻击病毒后导致更大的二次应答的证据。由于T02(BEI-VH)组中的多个样品在测试的最高稀释度(1:640)下具有可检测的中和抗体,这些结果可能代表组间差异的保守估计。

表19:猪死亡率和母猪血清学数据在下文概括。

PDCoV研究

该研究的目的是确定原型PDCoV疫苗在宿主动物中的功效。基于开发PDCoV的攻击材料和疾病模型的以下初步研究,未接种疫苗的仔猪在攻击后一周内显示出重量减轻和腹泻并且恢复。没有观察到死亡率。

表20:猪丁型冠状病毒(PDCoV)攻击材料:

为了建立关于猪丁型冠状病毒(PDCoV)的攻击毒株,在BI-ST细胞上生长两个内部攻击分离株(第3和4组),并且与NVSL PDCoV分离株(Illinois 2014)进行比较。基于它们所具有的突变及其与NVSL(Illinois 2014)分离株的遗传距离,选择第3组和第4组中的分离株。与NSVL PDCoV分离株相比,第3组病毒(BI PDCoV-5.0327)具有在刺突糖蛋白中的3个氨基酸缺失,以及在ORF1AB基因中的两个变化。与NVSL PDCoV相比,第4组病毒(BI PDCoV2.0307)具有在刺突糖蛋白中的4个氨基酸差异,以及在ORF1AB基因中的3个差异。

攻击的仔猪就腹泻进行得分,其中0=正常,1=异常(非纯液体)或2=攻击后的严重腹泻(纯液体粪便)。在阴性对照组中未观察到腹泻。另外,受NVSL PDCoV攻击的组在攻击后未显示任何临床体征、体重减轻、呕吐或腹泻。第3组和第4组的仔猪显示出严重的临床体征,其中100%经历严重腹泻(得分为2),伴有呕吐和体重减轻。另外,第3组和第4组中的母猪患有腹泻(攻击后D6-D8)并且进行治疗。先前公布的研究描述了感染有PDCoV的仔猪中的延长感染(和病毒脱落)。他们的观察结果与我们的结果一致。

NVSL PDCoV和阴性对照组中的动物在攻击后重量增加。相比之下,与对照组相比,用两种攻击分离株BI PDCoV-5.0327(第3组)和BI PDCoV 2.0307(第4组)攻击的动物在相同时期内显示出较不稳健的重量增加。

功效研究:

表21.PDCoV研究设计(疫苗组、剂量和途径)

对于功效研究,在分娩前5周和2周通过适当的途径用如表21中详述的每种疫苗制剂给四组母猪接种疫苗。在每次疫苗接种时在施用治疗之前,从母猪收集血清用于血清转换测定。在每次疫苗接种时从母猪收集粪便样品,并且测定疫苗脱落的证据。

来自免疫母猪的仔猪在分娩时入选试验。使用经口途径,在大约5至7日龄时,用1.0mL攻击材料接种仔猪。

从攻击后天数(DPC)0到DPC14,每天一次监测仔猪和母猪两者的临床体征。攻击后粪便和血清样品取自所有仔猪和母猪。测试粪便样品中PDCoV RNA的存在,并且测试血清中抗PDCoV抗体的存在。在分娩时或接近分娩时从所有母猪中收集初乳。在DPC3时以及在DPC10到DPC14之间的一个点,从所有母猪收集乳。测试初乳和乳中抗PDCoV抗体的存在。在DPC 0、7和21时监测仔猪的体重,而终末血清收集在DPC21时。

根据本公开内容,无需过度实验可以制备且实施本文公开且请求保护的所有组合物和方法。虽然已根据优选实施方案描述了本发明的组合物和方法,但对于本领域技术人员显而易见的是,可以对本文所述的组合物和方法以及方法的步骤或步骤顺序应用变化,而不背离本发明的概念、精神和范围。更具体而言,显而易见的是,化学和生理学两者相关的某些试剂可以取代本文所述的试剂,同时实现相同或相似的结果。对于本领域技术人员显而易见的所有此类相似的取代和修改被视为在如由所附权利要求限定的本发明的精神、范围和概念内。

下述参考文献在某种程度上提供了对本文所述那些的补充性示例性程序或其它细节,具体引入本文作为参考。

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序列表

<110> 勃林格殷格翰动物保健美国有限公司

<120> 猪冠状病毒疫苗

<130> 10-0176-WO-1

<150> 62/452026

<151> 2017-01-30

<160> 53

<170> PatentIn version 3.5

<210> 1

<211> 25505

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus Genome cDNA - NSVL

<400> 1

gcacctgaaa ccaagacgcg tcagtagtag ggactactgg tgcagccatg atagattggt 60

gtcggtagag aactagcgaa gctagggaga taaaattata gactaatgct ataattttta 120

tctttagtct ataattttat ctccctagct tcgctagttc tctaccgaca ccaatccagg 180

tgcgtctgcc accaagttgg ctacccttcc taggggcgct ttcgcgcttg ctcaccatta 240

gattacctgg aaaccagcca ttcaggttgg agtttcccca ggctcttttg tgtgggcatt 300

agcggcttgt ggtttttgca caaaatctaa gctacttacc gttcctctga ccatccacca 360

cttctataga cagcactgat taccgtaggg tttaagtcac accggtctgc accgcccgtc 420

agcggacaca ttacccagca tagcactcct tgcaccgagc ctaggtagga taaaaccccc 480

taccgggtga ctcttaaggc gtttcctcca cgggatagcc actagtcact aggtgtaagt 540

gatctgatct gggcgtattg tgttgcgcaa gtgtgatacc cataggagcg tggaatccta 600

ttctgcggct cagtgcctga tatagctgtg aaatggccaa gaacaagtcc aagcgcgacg 660

ctattgcgtt gcctgaaaat gtaccaccac ctctgcaact tttcattcat gttgcagctg 720

ctgaagaggg tcaccctaag gttactactt accttggcaa ctataacctc tatgccacca 780

aggctccgcc tggcgtgcag gttcttagtg ctaaaacctc tcttactgac tttgagaatg 840

tctttggagc tcaacccacc ttgcgatcaa ttcgtaatct ggtttgtgag gctcgctcgg 900

ctgaatggac aacttccaag aatgcttttg cactcaaagc cactcaactt gactactctg 960

atgccgtttt gagggcaatg attcgtttct gccctccaaa ggtgtccaca ctcgttgcct 1020

ttgctctttt tggccgattg gttaaaattg aggacaagga acttgctgag ttagctcgtg 1080

atactgccct tgagttggcg tacacggcta aaattggtac atctcttgct gacacgagat 1140

ctgtctcact tattcataag gacgcttatc taactctcag taatgaggtt gttggcgtaa 1200

cttttactgc cgcacttatg gcaaaggcta ccactgttaa tggagcaatg caatactcaa 1260

acttttacct ctaccctcgt gccactatta aggtaaccga tggtaaggct gaagcaattg 1320

caactaagcc tctgtctgct gccactaaag gcaagcaaat cacagaggat gtcaaccttc 1380

tccctgacta tcagcagctg cttgttgatc aagtgactgg cactgaggtt aaggttggag 1440

ctctaaccta tgttaagacc actgattcgc caccccttta ctttcccaaa gtcaagggtg 1500

gtgttattgg tattgcactt aagcagcagg gcactgcggc taagaagctc aatgtagtct 1560

tccatgctca acctgatgat gttctgctag ccttcataca acttcagcaa ttcttgaacc 1620

gtacttcgga ttcaagtgtt gaaattactg attgccagag ttatgaagta tctccaactg 1680

tgacggtcaa aattggcccg tctaaacctg gggatgtcat cgtggctact gatgaggaat 1740

accttaaatg ctttgaaacc cctgaggtag gtaggctcta taaggttttc caaactcaat 1800

cttgggctat cattgagcgt gccttctcca gtttgaagat ccgcgtgtcc aaagctttat 1860

cagcatttat aagttttctg caaaaccttg cagataactt tactgcaata agtggtgttg 1920

tcactgcact cattcgtgaa ctccaggatc ttaccctgga tgtggcgaca cgtatcacta 1980

acatacaatt tgtttaccgt gccggtaagc ttattgtcga cacgacaagt gtcatagcta 2040

aacttttcca gccattttgt gattttatat cacctttcct tcggaaagtt gctggttttg 2100

caatttacac tgttggtaat cgcatgctta tgtttaccag cactggcacc tttcttctca 2160

caaaggcaac tactaagata ctcaataagg caaagtacat ctttgatgtg gagcctgagt 2220

acccagtaga tgtaacaaca tccaaagttg tagtacatga agcactccag caaaccgaca 2280

ctaagcctac tagagctctg gaggctgttg atgtcgttgt tggtaatact gtactgcaaa 2340

tggctactga tggcactgcg ttctacccat cggatggtac gcacgcctct cttccaggat 2400

tcaaagcagg ttcggatgag cttttcataa gcttcaactg cgacctcttt gatgatgaga 2460

ctaatgctca aatcaacgaa atactcgctg catatgagct taaccaacta gtggctccag 2520

gtgattctac accgcgtcaa attgcgacgt tggttgtcga tacacttgca gatgctataa 2580

cagaccactt tccggagaaa accattgatc tacctgaaga ctatcaagtc ttttctgacc 2640

atgatgacct cccactcgca caataccaca tccctgatca cctgagcctg tatattcagg 2700

ctatggaagg tgaagatgat agtggtgatg aaatatgtat tgaggacgat gattacgact 2760

gtcctcaagc cgacgaagac acagaaggag taattcccca acagtgggaa cttcctgatg 2820

ttgataaatt tttactcaag atccaggaac ggaagaccag cagcgacgaa gtacttagcg 2880

tcgacgtcta tcctaaacca gagccggtcg gcaatgttgg gattgacgac agcgcgtcgg 2940

aaaagaagcc aaatggggac tcagtaccgg atcctgaggt ccatccaaca ctagagagtg 3000

tggatgttga acgaccaacc gaaacagcaa accaggctgt tgaagacaaa ccttctgata 3060

ccacctttgt ggttgatgag gaacaattac aagaatcaac accagaacat gaactccgct 3120

cctatgaagg ggagtttgat tctgatgatg aaattattat tcctatagta ccagtaacac 3180

ctgcggattt aaaaccacag actattacta taaaggagta ctttaagtct gaaaaacttg 3240

agactattaa cgaaggatcc acagagtcag ttacacaatc tgacgattcg tttgacgagt 3300

catttgttga tgctgagtct gatgatccac aagatcctgc tgtatatgat gatacaacaa 3360

ttataacgga cagcactgat gtaggcgatg agcctgagac aactctagct accatcgtta 3420

acacacctct gacactcgat aataacttgc cacctgaagc cattaaacaa cccagcccaa 3480

ctaaggttga gttagttgtt ggtgaattgg cgagtattaa atttgacaat tctgttctag 3540

tcaaccctgc taatgcgcaa ttaacaaatg gcggtggagc tgctcgtgca attgcaaaat 3600

tagctggtcc aaaatatcaa gagtactgta atagtgtggc tcctatctca ggaccgctta 3660

ccacggactc ttttgatgcc aagaaatttg gtgtagcctg catcttgcat gtagtgccac 3720

ccaaaggttc tgaccctaat gtacaagaac tcctgtatca agcttacaag agtatcctta 3780

ctgaaccagc acactatgtt atacctatac taggtgctgg tatctttgga tgcaacccag 3840

tccactctct ggatgcgttc aggaaagcat gtccaagtga cataggtcgt gtcacccttg 3900

tcactatgaa caaaaaccat ttgcaggtgt gggatgctct caataggacc attgtacgca 3960

ccactactga ctatgatcaa gttaccacca aggcccttac accccaggga gtgttagaag 4020

ccaatctctt tgatggtgag gactttgttc aagaaccaaa acccggtcaa atctaccttg 4080

aggttactga agaagttcag aaccaagcca aggaacttga ccttaacctt cagcaatact 4140

gcgtctacct gaagacttgc caccataaat gggttgtgag tcgtacgaac gggttgatgc 4200

atctaaaaca aaaagataac aattgttttg ttagtgcagg tgtaaacctg tttcaaaaca 4260

ctgcttatca acttagacct gctattgatg ctctctatag ggagtatctt aatggtaatc 4320

caaatagatt tgttgcttgg atctacgcat ccactaaccg tcgtgttggt gagatgggtt 4380

gtccacagca agttatttct ttgctcgtta gtaactctga cgcagcattt tcagcaacta 4440

cagcctgttg taacacctac tttaaccaca caggtgttat ttcagtagct cgtgaatatg 4500

acccaataca accaaaggtc tactgcatga agtgtgatgt gtggactccc tttacacccc 4560

agagtggaaa aggtgcagtt gcaattggta tttctgcaga tgaacctacc ggtcctgcca 4620

ttaaatttgc cgcagctcac tgctggtaca ctaatggcaa gaaaacagtt aatggctatg 4680

acactaaagc taatgttgta gctacctatc ataggtttga cgtgcctaag cctcaacttg 4740

tcgaggacgt ggttgcgctg cctactaaaa atgactttga agttctcaat gttgaagaac 4800

tgccgcagga tagtgtgctc catttggacc cacctcctgt acaggcctta caacctaagg 4860

ctaaccaaca cattgagatt ctagaaaacc cagattatct ggacattttg gatctttgga 4920

ttcgtaaacc caaattcatc ctcgtaaagt cgtggagtgt tttgggtaga gcactatgta 4980

aggcaggtaa agttgtcttt gtcaatgctt cgcttttgac ccgtttctac aattaccttg 5040

tagagattgg tgctcttgac tcaacaataa ggttgtcagt cgatcttacc tgtaaatttg 5100

ttagaacggt tctcccatcg tctaacactg tacacaaaac ttgtcttggt ctgtattatt 5160

cagcccagac actttttgtt tctttagcac cattccttat gttaccagct gtagttagtc 5220

tgcttaattc aggctataca attggcacat atttgtatgc aaaaactggc tggccttgta 5280

attacaatgc cacgcaacac tttgattata attcttactg tgcaggtgac ttggtttgtc 5340

aagcctgttt tgacggtcaa gactccctac atttgtatcc gcatttacgt gttaatcagc 5400

agccccttca gaccactgac tacactgttt atgcgctttc actaatacta ctattagcta 5460

acatgactct tgtcatgggc acgctaatag ttactttctt tgtgaacttc tatggtgtgc 5520

aaataccatt ttatggtaca cttttgatag attatcaatc cgcactggtg attactttct 5580

cagtgtacta cttttataag gtaatgaagt ttttccgcca tctcacacat ggatgtaaaa 5640

ttccaacgtg tgtggtatgt gccaaacttc gtaccccacc tactataaca gttgagactg 5700

tcgttcaggg caggaaatac ccatctgtta ttgaaacaaa tggcgggttt acaatttgta 5760

aagaacacaa cttctattgc aaggactgct ctttacaaac acccggcact ttcatcccga 5820

cagaagctat tgagtcgctc tcacgagcta ccaggcttag tgtcaaacca acagcaccag 5880

cattcttact tgctagagat gttgagtgcc aaactgatgt tgtcgttgct cgcgcaatgc 5940

ataaccaaaa tgcgcatgtg tgcatttcaa aatactctga tatccgtacc gttgaccaac 6000

tacttaagcc tactccactg ttttcataca ctcccgatgt tatcatcgcg gcagactttg 6060

acaacagagg tagtcttaag acagctaaag aattagctgt ggttttgtca atggacctta 6120

aacgtactat aattatcatt gatcaggcct attctagacc tattgataat tatcaggaag 6180

ttgcttctcg tattgagaag tattacccag ttgcaaagat cacacccaca ggtgacatct 6240

ttacagacat taagcaagcg accaatggcc aagctagtga ctctgctatt aatgcagctg 6300

ttctggctgt ccagcgcggt cttgatttta caattgacaa ccctaacaac atattaccac 6360

attacgcctt tgacttttca accctcaatg cagaagacca gtctaccatt ttggagagtg 6420

gttgtgctaa aggcaatctc aagggcacta atgttggtgt tgttctttca gctagccttg 6480

ttacacgtct tagtcagcag gctatacgtg tgattgctaa tgctgcttca cgtaatggtg 6540

ttacatgcgc tgttactcct tctacacttg ttatgcgtgg gaatattgca acacagccct 6600

tgactcgcat caaagctggt gcacctccca tgcgtcaaaa aattttatgt gttatcctgg 6660

cacttgctat tgtgtacttt gctgctatgg cttttggctt tttggcaagt caacttacgc 6720

ttaatacagt gcctacgatt aaatctgata tccgcgcctc taccttctac gttgttagag 6780

atggagtctt ggatactgtt cgttcaaatg acaagtgctt tgcaaataag tttttggcat 6840

ttgatagctt cattcaagca ccttacacta attcacctga ctgtccagtt gttgtgggag 6900

ttgttgatgt aacgacgcac tctattcctg gaattccagc aggtgtcatt catagagacg 6960

gtctcatact taacatttat gaacagtctc tttatgaaac tcatcagcgt cagtctatgg 7020

ttagggatgc gttgtcactc aagacagcaa atctctttaa cctaggcaag cgtgttgtag 7080

taggatacac tcaacatgaa gttgttgtgg gtacctccta ttttaattct cctgcacttt 7140

ttaatgcaaa gtgcaccttc ttacagtatc aggacactag acaactctat tgctatgata 7200

ctgttcctac tgaacataag ctctactctg atgtgcttcc gcacgtcgag tataaggcta 7260

ttgacattaa tggtgatctt gttcctttca agataccaga gcagataatg ttctatccac 7320

atattgtgcg ctatactagc aattcctatt gccgtatggg gcattgtttt aatactaacc 7380

ctggtatttg catttcattt acggacgaat ttccgtatag tgaaaatgtc aaacctggtg 7440

tgtactgtgc tgatacctct ttgcagttgt tttcaaacct cgttttgggc actgtatctg 7500

gtattcacat ctttacatca acagctgcat tgcttggatc tactattgtg atcatactat 7560

gcgttgttgc tgttcttgca gttcagcgat tcttcaagga gtacacaact tttgttatgt 7620

acacttgtgg tcttgctctt gtcaacattg taggcattgc acttatgtac aagtgccttg 7680

tcttcgcgat tttctattat gcaatctacc tttactttgt ccttactttc ccctccttta 7740

agaggaatgt ggcattgttt tacttcgctg tagtgatcgt gccgcacgtg agtaacatgc 7800

aattgcttgc gctcattgtg tgtagcatta tctactttct ctacacctat gttcatactg 7860

tagctaagac agctgggaaa ttttcttcct tcttagacgc agctaaagct acttttgtca 7920

ttgacaatga aaagtacgtg ttgcttaaag acctcgctgg tgctgaattt gaccagtatc 7980

tggcctctta caacaagtac aaatattttt ctggtactgc ttctgataag gattatgata 8040

aggtctgtat ggcatttctt gccaaggctt tgtcatcttt tcgtgaagga ggcggttcac 8100

agttgtacac accacctaaa tttgcagttg ttcagagtct taagaccaag ctgcaagcag 8160

gtatcaaaat cctcctgcac ccttcaggtg tagttgagcg atgtatggtc tcagttgtct 8220

acaatggatc tgcattgaat ggcatctggc ttaagaatgt tgtctactgc ccacgccatg 8280

taattggaaa attccgtggt gaccagtgga ctcacatggt ctcaattgct gattgccgcg 8340

actttatagt caagtgtcca atacagggta ttcagctaaa tgtccaatca gttaagatgg 8400

taggagctct cctccagtta actgttcata ccaacaacac agccactcca gactataagt 8460

ttgaaaggct acaaccagga tcatcgatga caattgcttg tgcttatgat ggcattgtac 8520

ggcatgtcta tcacgtggtc ctccaactta ataatcttat ttatgcaagc ttccttaacg 8580

gagcttgtgg tagtgtgggt tacactctta agggtaaaac actctactta cattacatgc 8640

accacattga gtttaacaac aaaactcata gtggtacaga tcttgaaggt aacttctatg 8700

gcccctatgt ggatgaggaa gttattcagc aacaaacagc attccagtat tacactgata 8760

atgttgttgc tcaattatat gcacacttac tgactgttga tgctagacca aaatggctgg 8820

cacaatctca gataagtatc gaggatttca actcatgggc tgctaacaat tcctttgcta 8880

acttcccatg tgaacaaact aatatgtcct acattatggg actctcgcaa acagctcgag 8940

tccctgtaga acgtatcctc aataccatta tacagctaac caccaataga gatggtgctt 9000

gtattatggg atcttatgat ttcgagtgcg attggacgcc agagatggta tacaatcagg 9060

ctccaatttc attgcagtca ggagtagtta agaaaacttg tacgtggttc ttccacttct 9120

tgtttatggc tattaccatg ctactcgctg ccatgcatgt tttccctgta cacttgtatc 9180

caatagtact gccatgcttc actgtcgtgg cattcctgtt gactttaacc attaaacaca 9240

ctgttgtgtt taccactaca tacttgcttc cgtcactttt gatgatggtt gtaaatgcta 9300

acactttttg gataccgaac acatttctgc gcacctgcta cgaaactata ttcggttccc 9360

caattgctca gcgactgtat ggttacactg ttgctcttta tatgctgatc tatgctggac 9420

ttgcaatcaa ctatacgttg aaaacactcc ggtatagagc aacttcattc ttatcttttt 9480

gcatgcagtg gtttcaatat ggttatgttg cacacattgc gtacaaactg cttaataaac 9540

cctggacaga atcactactc ttcacagcct tcacaatgct aaccagtcat cccttgttgg 9600

ctgctcttag ctggtggcta gctggtcgcg taactctgcc cattatcatg cctgacttag 9660

ctattcgtgt tttggcgtat aacgtcattg gctatgtcat atgtgttcga tttggcctta 9720

tgtggcttgc aaatcggttc acaactgtac ctatgggcac ataccagtat atggtgtctg 9780

tagagcaact taagtacatg atggcagtta agatgtcccc accgcgtaat gcgtttgagg 9840

tgcttatagc caacattaga cttcttggtt tgggtggaaa ccgtaacatt gctgtttcta 9900

ctgtccaaaa caaaattctt gatgcaaaag ctactgctgt tgttgttgct aaccttcttg 9960

aaaaggctgg cgtcacaaac aagcacgcta tttgcaaaaa gattgtgaaa ctccacaatg 10020

atacccttaa agccaccact tatgaggagg ttgaggtagc acttgtgaaa cttctttctc 10080

acataattga gttcttgcca actgatcagg tagatgctta tctagctgat gcggccaatg 10140

ctcaacatgt taatacctat ttagacaact tgcttgagaa caaagctgtt gttcaggctg 10200

ttgccgatat caacattaat ctggattctt atagaattta taaggaggca gatgctattt 10260

ataaacgatc tgttgagatg aacgaatctc cgcaggagca aaagaaaaag cttaaagctg 10320

ttaacattgc aaaggcggaa tgggagcgtg aggctgcttc tcagcgtaag cttgaaaagc 10380

ttgctgatgc tgctatgaag tctatgtatc ttgcagaacg tgctgaggat cgtcgcatta 10440

agctaacctc tggacttact gcaatgcttt accatatgct tagacgtctt gactcagata 10500

gggtaaaagc tctgtttgag tgcgctaagg cacaaatctt gccaatacat gctgtagtcg 10560

gaatttctaa tgacaacctt aaagttattt ttaacgataa ggacagctac tctcattatg 10620

tagagggcaa cacacttata cataagggag ttcgctacac tattgtgaag aaactctcct 10680

tagataatgc acctattgaa ggcgtaccag aagaattccc tgtggtcgtt gagactgtta 10740

gggaaggtgt gccccagttg caaaataatg agctatgttt gcgcaatgtt ttcactgctc 10800

agaacacagc tcaggacttc aatggcaatg aatccactgt aaaatctttt tatgttacta 10860

gaaccggtaa gaagattttg gttgccatta catcaactaa agacaatctt aagactgtga 10920

cctgccttac tgagaccggt aagacagtcc ttaacttgga cccccctatg cgcttcacac 10980

ataccgtagg tggaaaacag tctgttgtct atctctattt tattcagaat attagttcac 11040

tcaacagagg tatggttatt ggccacatct ctgaaactac tatccttcag gcaagtggca 11100

ctcaaattga gtaccagcaa aatgcctctc ttttgaccta tttggctttc gctgtagacc 11160

ctaagacagc ctaccttaag catcttgctg atggtgggtc tcctatacag ggttgtattc 11220

agatgattgc tactatgggt cctggatttg cagttactac taaaccacaa cctaatgagc 11280

atcagtattc ttatggtggt gcttcaattt gtctttattg ccgtgctcat ataccacatc 11340

ctggtgttga tggacggtgc ccctacaaag gccgctttgt tcacatcgac aaagataagg 11400

aacctgtttc cttcgctttg actcatgagc catgcagttc ttgtcaacgg tgggttaatt 11460

atgactgcac ctgcggatct agtctgcaga attcggctta tttaaacgcg taacgggttc 11520

tagtgacgcc cggctagaac ccctgcagcc tggaactcaa ccagatgctg taaaaagggc 11580

cttccatgtg cataatgata ccacctctgg tatattctta agcacaaaat ctaactgcgc 11640

tcggtttaaa accacacgca gtgccctgcc tttacctaat aagggagagg ttgaattgta 11700

ctttgttact aagcagtgtg cagctaaagt cttcgaaatc gaggaggaat gctacaacgc 11760

tcttagtaca gagctttata ctactgatga tacatttggt gtccttgcca aaactgagtt 11820

ctttaagttt gacaagatac ctaatgtcaa tcgccagtat ctgactaaat atacactcct 11880

ggacttggct tatgctctac gtcatttgtc aacatctaag gatgttattc aagaaatctt 11940

gatcaccatg tgcggaaccc ctgaagattg gtttggggaa aattggtttg atccaattga 12000

gaacccatcc ttttacaagg agttccataa acttggggat attcttaacc gttgtgttct 12060

taatgccaat aagtttgcta gtgcctgtat agacgctggt cttgttggca tattaacacc 12120

cgacaaccaa gacctcctgg gtcagatcta tgactttgga gattttatta ttacacaacc 12180

aggtaatgga tgtgtggact tagcatccta ttattcttat ttaatgccca ttatgtccat 12240

gactcacatg ttaaagtgtg agtgtatgga tagtgatggc aacccacttg agtatgatgg 12300

atttcagtat gacttcacgg acttcaagct tggcttgttc gagaagtatt ttaagtactg 12360

ggaccgtcct tatcatccta acactgttga atgtccagat gaccgttgcg tattgcactg 12420

tgcgaacttc aatgtgttgt ttgctatgtg tatacctaat acggcatttg gcaatctttg 12480

ttcaagagct actgttgatg gccaccttgt ggtccagaca gtgggtgtac acttgaaaga 12540

actcggtata gtccttaacc aggacgttac cacacacatg gcaaatatta atctaaacac 12600

tctattgcga ttggttggtg atcccaccac tattgcaagt gtctcagaca agtgtgtaga 12660

tttaagaact ccttgtcaga ccttggctac tatgtctagc ggaattgcta aacagtcagt 12720

caagcccggg cattttaatc aacacttcta caagcatttg cttgatagta acctattaga 12780

ccaacttgga atagacattc gccacttcta ctatatgcag gatggtgaag cggctatcac 12840

agactacagc tactacaggt ataatacccc cacgatggta gatatcaaga tgttcttatt 12900

ttgccttgag gtggcagata agtatcttga gccctacgaa ggtggatgta ttaatgcaca 12960

gtcagttgtg gtctctaatt tggacaagtc agcgggctac ccctttaaca agctaggtaa 13020

ggctcgtaac tattacgaca tgactcatgc cgagcaaaat caactgtttg agtatacaaa 13080

acgcaatgtt ttgcctacac tcactcagat gaaccttaag tatgcaattt cagccaagga 13140

tcgtgctcgc actgtggcag gagtgtctat aattagcacc atgactaaca ggcagtacca 13200

tcaaaagatg ctgaaatcta tttcacttgc acgcaatcag accatcgtga ttggaacaac 13260

caaattctat ggtggttggg acaacatgtt acgacgactg atgtgtaata tcaacaatcc 13320

cattttagtg ggttgggatt accctaagtg tgatcgttct atgccaaaca tgctgcgcat 13380

tgccgcttcg tgcttgctag cacgaaaaca cacttgctgt aaccaaagcc agcgattcta 13440

ccgtttggct aatgaatgtt gccaagtact atctgaagtg gtagtctctg gtaacaacct 13500

ctatgtaaaa ccaggtggca ctagcagtgg tgatgcaacc acagcttatg ccaactcggt 13560

atttaacatc ttacaggtgg tttctgctaa tgtagccacc ttcttatcaa cttccaccac 13620

gacacatctt aataaggaca ttgcggactt gcatcgtagt ctttatgaag atatttatcg 13680

tggtgactct aatgatatca ccgtcatcaa tagattctac cagcatctcc aaagttactt 13740

tggacttatg atattgtctg atgatggtgt cgcatgcata gactcagccg ttgcaaaggc 13800

tggagctgtt gctgatcttg atggtttccg agacattttg ttttaccaaa acaatgttta 13860

catggcagac tcaaagtgtt ggacagaaac tgacatgaat gttggccctc atgaattttg 13920

ctcacagcat actgtgttag cagagcatga tggtaaacct tactacttac cttacccaga 13980

tgtctctcgc attctgggtg catgcatctt tgtggatgac gttaacaagg ctgaccctgt 14040

tcagaacctt gaacgttaca tctcacttgc aattgatgca tatcccctca ccaaggttga 14100

ccctattaag ggtaaagtct tttatttgtt actagactac atacgtgttc ttgytcagga 14160

gttacaggac ggtatccttg atgctttcca atcactcact gacatgtcgt atgtaaataa 14220

ctttatgaat gaggcctttt atgctcagat gtatgagcaa agtcctacac tacaggccag 14280

cggtgtttgt gtggtgtgta attcacccac tatactgcgc tgtggtgatt gcattcgtcg 14340

accactactt tgttgcgtct gtgcctacca gcatgttacg cagactacac ataaacgtat 14400

cattgctatc aacaactaca tctgtagtgt tgagaattgc aatgaggaca atgttgaaaa 14460

acttttcatt tctggcactg cgatttattg tgagaatcac aaacccacgc tgtgcatacc 14520

cattgtagct aatggttctg tttttggtat ctatcgccac actgcccgtg gtagtgatga 14580

catagacctc tttaacgagc ttgctacatc taactatgac actattgaac cttatcagaa 14640

ggccaatcgt gcacctttat cacttatgct cttcgctgct gaaaccatta aggcactcga 14700

ggagtctatc aagaagtcat atgctaccgc aaccgtcaag gatgtgtatg accaacgctt 14760

cattaaactt ctatgggaac agggtaaaaa gccgccaccc ataacgaaga accacatttt 14820

cactggctac cattttaaca agaatggaaa aacccaagtt ggtgattaca ttcttgctaa 14880

aacagatggc agtgacactt atacttacag aggaacatct acctacaaac tccaaacagg 14940

tgatgttcta gtcttaatgg cacatgttgt tacaccgctc tcagcacccc ctgtgttaac 15000

gcagacaaca tatgtcagaa aatcactttt acccgactct gttggtgcgt cttattatgt 15060

gcaacatttt aagtcatata atgagatagc tatgcagagg gttacaacag tattaggtcc 15120

accaggcaca ggtaagtcaa cctttgctat tggtttggct aagtactttc ccagtgcacg 15180

tatttgctac actgcgtctt cgcatgcagc aatcgatgca ctctgtgaaa aagctttcaa 15240

gacaatacct gtaggccaat gcagtcgtat cgtacccaca cgtacaactg ttgagtgctt 15300

tcaggagttt gtcgtaaata acacaactgc acagtatatc ttctcgacta tcaatgcctt 15360

acctgacatt aagtgtgaca ttgtagttgt agatgaggtt tctatgttga ccaattatga 15420

gctttcctct gtgaatgctc gtttggttta caatcacatt gtgtatgttg gtgatcctta 15480

tcagttacct tcacctagaa ctatgcttac gtctggccag ctttcgccag ctgactataa 15540

cgtagttact gatataatgg tacatgcagg agcggatgtt atgctcgaca tgtgctacag 15600

atgcccacgt gaaatcgttg agacagtgtc taaacttgtc tacgataaca aactaaaagc 15660

ggcgaaaccg aactcaagac agtgttacaa gaccattgtg aactttggtc ctggagacgt 15720

tgctcatgag ggacaatctg cctacaacga agcacagttg cgtttcgcac tcgcatttag 15780

acaacaaaag cggtgggata acgtgacttt catatctcca tataatgcta tgaatgtgaa 15840

agcatcctta gcaggtttct ctactcagac cgttgactct tctcaaggtt ctgagtatga 15900

ttatgttatc ttttgcgtga ccactgattc agcacacgca cttaacatgg ctcgtttgaa 15960

cgttgccctt acacgcgcaa agataggtat ccttgtggtg tttaggcagg caaacgaact 16020

ttacaatagt ttgcagtttg aatctattga ttcacagctt cagtcgagtg ctgagaaaaa 16080

cctcacacca ctgtttaagc gctgcggcta tgagtataat ggcgtccatc cagctcatgc 16140

tttgacctgg catgattgtg gtgcagagta ccgctgtgag gagccacttg ctaaattagt 16200

aggagttgcc gatggcactc ttatatcata caaaacccta gtatccacac ttgggtttct 16260

tccatcactt aaaattgatg catatcataa tatgttccta acacgtgacg cgtgtcgcac 16320

ctatgttcag agttggatcg gcatagatgt tgaagcagca cacgccataa aacctaacac 16380

cgggactaac ctgccattgc aaataggttt tagtaccgga aagaattttt cagtcactcc 16440

agagggaatt tgggtaaacg agcacggatc ttgcactgag cccgtccctg ccaaaatacc 16500

tcctggagaa caatttcgtc accttaaaaa ggacatgcgc caggcgcgtc cttggaaggt 16560

tgttcgacgt gagattgcta ctcacattgc tgaggtagct cctcacactg attatatatg 16620

ctttgtcact tgggctcacc agcttgagct agcgacaatg cgctactttg tcaaactagg 16680

tatggaagag aaatgctttt gtggcaggcg ggcttgtttc actaatggaa ctgagttcgc 16740

ttgcaaagca caccattctc tcaccattcc acaatgtgat tatgtgtaca atccattcct 16800

catcgacgtg gctacgtggg gattctcggg acggctttcc accaaccatg acgctgtgtg 16860

cacatatcat gctaatgccc atgttgcatc agctgatgca atcatgacgg tatgtttagc 16920

tatccatgaa ctgttcagta ctgttgactg gaactttgaa tttccagtaa ctgctgagca 16980

atcgcaactt aacaaggcct gtcgcttagt acaggcgaat tacttaaata tactactcac 17040

tacaaccaaa gccacggtgg ttcacgatat tggtaaccca aaaggtatcc ctatcgtgcg 17100

caaacctggt gttaaatatc acttctatga tcaagcaccc attgtcaaac acgttcaaaa 17160

actaaagtac aagccagaga tggaggcccg tttcaccgat ggtttgacta tgttttggaa 17220

ttgtaatgtt gacacatacc ctgctaacgc ccttgtgtgc cgctacgaca ctcatcggca 17280

gaagcattta attggaccta atggttcagc actatatgtt aataagcatg cttttctcac 17340

ccctgagatg catacttatg ctacacataa actcaacttg gctccactca tctactactc 17400

caccacagat tgtagtagtg aacagcctat tgttgttacc tacagagatt gtgtcacccg 17460

gtgcaatact ggaaaaactc tctgtccaaa tcatgctctt gaataccaag agtttatcaa 17520

tgcatacaat ctcatggctc gccatggatt taatgtttac ataccacgca atgtcaacgt 17580

ttacaactgt tggcttactt tcactaatct ccaaaacctt gaaaacttag cttacaactg 17640

ttattataag aactgcaatg ctcacgttga tgggcagctt gatgtagtta ttaataataa 17700

cgctgtatat gctaaggtcg acaataatct tgtcaaactt ttcgacaacc gcactaactt 17760

acctgtctca gtggcctttg aacattacac taacaggcat acccgttcac tgccaactac 17820

acagctgtta tctggtttag gcgtaaccgc caccagaaat ttcactgtgt ggttcgacaa 17880

tgatacaatt ttccaataca ctattaatgt atctacgtat actgacatcg accctagtac 17940

ccatgttgtc ctctgtgatg ataggtacgg aacagattgg agtcagttta accaacttcc 18000

taatgcagta ttcctcacca aaactaaggt gaagaaaaca gaaccgtttg tttgtacagc 18060

actgacccta aatggcgtcg ccattgacgg tgaagagcta tacatctatg tacgctataa 18120

caatcaactg accacatttg ctactacttg tacacagggt agaaatgttg agcagtttat 18180

acctaaaaca cctatggaaa gagacttcct tgagatgtct caacagtcct tcatccagca 18240

acatcaattg caggaactgg gtgttgaaca cattatctat ggtgatgatt ccagtccagt 18300

cattggcgga actcacacac ttatctcact agttaaaaac aagtttgaac atcagcttgt 18360

caaccatgtt tacaacccag tccagaactg tgttgttacc tcacctaacg caagctccaa 18420

gaacgtttgc actgttcttg atgttcttct tgatgactac attgacatca taagacaagc 18480

acatgccagt tacacaagta aatccaaagt attcactgtg tcaattgaca accaacaaat 18540

tagattcatg ctttggcatg atgagcaagt caagacttgc tacccaatct tacagtcact 18600

taccaatggt taccagatgc catctgtgta caaaacattg gttactgact tacaaccagc 18660

tgacatccct aattatcatt cctacacccc ccgggtgcct ggagtagtta agaatgttat 18720

caagtaccgc caacttttca actacatagt taaaaaggat aggttggcag taccacacaa 18780

tatgaccgta ttacaccttg gagctgcatc tgcactaggt acagcaccag gttcttcagt 18840

cataaaacaa atgtttcctg aaggaactgt tcttattgac ctcgatataa gagagttcac 18900

ttcagatgct aaccaaataa tagttacaga ctacagaact tacataccac cacaccacgt 18960

agacgtcata ttttctgacc tctactgttg tgatgacata cacttctttg acaatctaat 19020

aaggatagtt aaggagaggc tcgccctcgg tggttctatc tttgttaaga taactgaaca 19080

ttcattctca cccgaactct actcacttgc gggttggttc gatgattatc aactattttg 19140

cacagcagtc aatgcctcgt cttcagaagc atttttatgc tgttttaatt atttggggct 19200

tgctaaggaa aacattaatg gttttaactt acatgcttcc tatattcaat ggcgcaatga 19260

aatagcgttg acaccaacct attctccttt agcggacaac ccggctacgg cctgtaagct 19320

aaaagcaacg cctattatct cggctcgtga gttagagaag aagcctattc ttcgctatct 19380

cgttgcatca gggcgccttc ttgtgaggcc accagaatgc agagagctct attgattatg 19440

accttatttt gtctcgttcg agcaaagttt gctgatgatc tactcgattt gctcaccttc 19500

ccgggtgcac atcgcttctt acataaaccc acgaggaatt ccagcagtct ctactcgcgg 19560

gctaataata attttgatgt tggcgttctt cctggctacc ccactaagaa cgttaacctc 19620

ttctcaccac ttactaactc cactttgccc attaatggcc ttcatcggag ttaccaacca 19680

ctcatgctga attgtcttac taaaataact aaccacactc tcagcatgta tctcctacct 19740

agtgagatac aaacttatag ctgcggcggt gccatggtta aataccagac acatgatgca 19800

gttcgtatca ttttagacct cactgccact gaccacatct ctgttgaagt cgttggccaa 19860

catggtgaaa attatgtgtt tgtttgcagt gagcagtcta cctacaccac tgcattacac 19920

aaatctacct tcttctcact taattctgag ctttattgct ttactaataa cacctactta 19980

ggtattcttc cacctgattt aactgacttt acggtctacc gtactggtca gttctatgct 20040

aatggttacc ttttaggtac tttacctatt acggttaact atgttaggtt gtatcggggt 20100

catttgtctg ccaatagtgc ccactttgcc cttgcaaacc taaccgatac actcataaca 20160

cttaccaata ctactatatc gcaaatcact tattgtgata agtctgtagt tgattcaata 20220

gcatgccagc gctcttctca cgaagtggag gatgggtttt actccaaccc taaatctgcc 20280

gttagagcta ggcaacgtac tattgttaca ctacctaagc tccctgagct tgaagtagtg 20340

cagttaaata tttctgcaca catggatttt ggcgaagcca gacttgacag cgttaccatc 20400

aatggtaaca catcctattg tgtcactaag ccttacttca ggcttgaaac taactttatg 20460

tgtacaggtt gcactatgaa tctgcgcact gatacctgta gttttgacct gtcagcagta 20520

aacaatggca tgtcattctc tcaattctgt ctaagcactg aatctggtgc ttgtgagatg 20580

aaaattattg ttacctacgt atggaattac ttgctaaggc agcgtttgta tgttactgct 20640

gtagagggcc agactcacac tggaaccact tcagtacatg caacagacac ttctagtgta 20700

atcactgatg tctgcactga ctacactatc tatggagtct ctggtactgg cattattaag 20760

ccatcagatc tcttattgca caatggcata gtattcacct ctccaacagg tgagctttat 20820

gcatttaaaa atataaccac tggcaaaacc cttcaggtct taccgtgtga aaccccttct 20880

caactgattg tgataaacaa caccgttgtc ggtgctatca catccagtaa ttcaactgaa 20940

aataataggt ttactactac tattgtcaca cctactttct tttattccac aaatgccacc 21000

actttcaact gcactaagcc tgttttgtcc tatggaccta tcagcgtgtg tagtgatggt 21060

gcaattgtgg gaacatccac attacagaat actcgaccat ccatagtttc actatacgat 21120

ggcgaagttg aaataccatc tgcattttct ctttccgttc agacggagta cttgcaagtt 21180

caagcagagc aagttatagt tgattgtcct cagtatgtat gcaatggcaa cagccgttgt 21240

ctacaattac tggcacaata cacctcagct tgctctaaga ttgaagcagc tctgcattcc 21300

tctgcacagt tggatagcag agagattata aatatgtttc aaacatcaac acagtccttg 21360

cagttagcta atattaccaa cttcaagggt gactacaatt ttagcagcat actaaccacc 21420

agaattggtg gcagatctgc tattgaagac cttcttttta ataaagttgt tactagtggc 21480

cttggcactg ttgatcagga ctacaaatcc tgctctagag acatggccat cgctgactta 21540

gtttgttccc agtattacaa tggcatcatg gttctacctg gtgttgttga tgctgagaaa 21600

atggcaatgt atactggctc tcttactgga gctatggtat ttggaggact gactgccgca 21660

gcggcaatac catttgccac ggcagtacaa gctcgcctca attatgtcgc actgcaaaca 21720

aatgtactac aagaaaacca gaaaattctt gcagaatcat ttaaccaagc agttggcaat 21780

atatcacttg cactatcttc tgttaatgat gccatccagc aaacttctga ggctcttaac 21840

accgtagcta ttgctattaa aaagattcaa acagttgtta accagcaggg cgaggcatta 21900

tcacacctga ctgcacagct gtcaaacaat tttcaagcaa tttcgacttc tattcaagac 21960

atttacaacc gtcttgagga agtagaggct aaccagcaag ttgaccgtct catcacagga 22020

cggttggctg cacttaatgc atatgttact cagttactca atcagatgtc tcagattaga 22080

caatctcgat tgttagctca gcaaaagatt aatgagtgtg tcaaatcaca gtcgtccaga 22140

tacggtttct gtggaaatgg cacacacatc ttctcactta cacagactgc accaaatggc 22200

atatttttca tgcatgcagt gctagtaccc aacaaattca cacgtgtcaa cgcttctgcc 22260

ggcatttgtg tggataatac gagaggctac tcattgcagc ctcaacttat actctaccag 22320

tttaataact cctggagagt tacacctaga aatatgtatg aacccagact gccccggcag 22380

gctgatttca tacaattaac tgattgcagc gttacttttt acaacaccac cgctgctaat 22440

cttcccaata ttatccctga cattatagat gtcaatcaaa cagtcagtga tattattgac 22500

aatttaccta cagcaacacc tcctcagtgg gatgttggta tctataacaa cactattctc 22560

aacctcaccg ttgagattaa tgatctacaa gagcggtcta aaaacctctc acagattgca 22620

gatcgtttac aaaattatat tgacaatctt aacaatactc tagttgacct tgaatggctc 22680

aacagagtgg aaacttacct taaatggccg tggtatatat ggcttgccat tgccctggct 22740

cttattgcat ttgtgacaat cctcataaca atctttcttt gtactggttg ttgtggtggt 22800

tgctttggtt gttgtggcgg ttgttttggc cttttctcta agaagaaaag gtataccgac 22860

gaccaaccaa caccgtcctt taagtttaag gaatggtagt cgacgactgg gccgttacca 22920

tccctggaca atatattatt gctatactag ttgtcatctg cattggtgtg gcactacttt 22980

ttattaatac ttgcttagct tgtgttaaat tattttacaa gtgctaccta ggggcagcat 23040

atcttgttag gcctattata gtgtactact ccaagccgaa ccccgtacct gaggatgagt 23100

ttgtaaaagt acaccaattt cctagaaaca ctcactatgt ctgacgcaga agagtggcaa 23160

attattgttt tcattgcgat catatgggcg cttggcgtca tcctccaggg aggctatgcc 23220

acgcgtaatc gtgtgatcta tgttattaaa cttattctgc tttggctgct ccaacccttc 23280

accctagtgg tgaccatttg gaccgcagtc gacagatcat ctaagaagga cgcagttttc 23340

attgtgtcca taatttttgc cgtactgacc ttcatatcct gggccaagta ctggtatgac 23400

tcaattcgtt tattaatgaa aaccagatct gcatgggcac tctcacctga gagtagactc 23460

cttgcaggga ttatggatcc aatgggtaca tggaggtgca ttcccattga ccacatggct 23520

ccaattctca caccagtcgt taagcatggc aagctcaagc tacatgggca agagctggcc 23580

aatggcatat cagtcagaaa tccgccacag gatatggtga tagtgtcacc aagtgacacc 23640

tttcactaca cttttaagaa acctgtggaa tcaaacaacg atccagaatt tgctgttctg 23700

atataccagg gtgaccgcgc ttcaaacgct ggacttcaca ccataaccac ttcaaaggcc 23760

ggtgacgctc gcctgtataa gtatatgtaa tgtgcaactg ccatctgcag ctgcgagatt 23820

tatatagatt gtgcaataag cggcacatca gaagagagga tgttcctgag cttattgacc 23880

ctctcgttaa aactcgctgt tttgcttaca gtctcgtggt tcttgctaat gctaatccaa 23940

ttgcatttag catactacct cggaaaattc ttatcaatgg tgagccttta ctgcttgaat 24000

atggtagcat atatggtaaa gactttatca ttcgaccatc gctccaagtc attcttgaag 24060

atgaattaaa ttaaagtttt gacaccaatc tatcatggct gcaccagtag tccctactac 24120

tgacgcgtct tggtttcagg tgctcaaagc tcaaaacaaa aaggctactc atcctcagtt 24180

tcgtggcaat ggagttccgc ttaactccgc catcaaaccc gttgaaaacc atggctactg 24240

gctgcgttac accagacaaa agccaggtgg tactcccatt cctccatcct atgcctttta 24300

ttatactggc acaggtccca gaggaaatct taagtatggt gaactccctc ctaatgatac 24360

cccagcaacc actcgtgtta cttgggttaa gggttcggga gctgacactt ctattaaacc 24420

tcatgttgcc aaacgcaacc ccaacaatcc taaacatcag ctgctacctc tccgattccc 24480

aaccggagat ggcccagctc aaggtttcag agttgacccc ttcaacgcta gaggaagacc 24540

tcaggagcgt ggaagtggcc caagatctca atctgttaac tccagaggca caggcaatca 24600

gcccaggaaa cgcgaccaat ctgcaccagc tgcggtacgt cgtaagaccc agcatcaagc 24660

tcccaagcgg actttaccca agggtaaaac catttctcag gtatttggca accggtctcg 24720

tactggtgcc aatgtcggct ctgcagacac tgagaagacg ggtatggctg atcctcgcat 24780

catggctcta gccagacatg tgcctggtgt tcaggaaatg ctttttgctg gccaccttga 24840

gagcaacttt caggcggggg caattaccct taccttctcc tactcaatca cagtcaagga 24900

gggttctcct gactatgaga gacttaagga tgcgctcaat acggtcgtta accagaccta 24960

tgagccacct accaaaccaa ctaaggacaa gaagcctgac aaacaagacc agtctgctaa 25020

acccaaacag cagaagaaac ctaaaaaggt aactctgcca gcagacaaac aggattggga 25080

gtgggatgat gcttttgaga taaagcagga atcagcagcg tagacatcaa tctatgtctg 25140

ttaaacccac ccaactccac tcaaatatct ctttggttcc agagagtcgt agtgtatagc 25200

cagagagcca gtcagagggc gctatcatgc aaactagggc tggctactct agcacagaat 25260

cacatcccga taatcaacag tgctagaagg ttgattatac catttaatat gccgaggcca 25320

cgcggagtac gatcgagggt acagcataat ctcaactttt gttgagccac aattttaatc 25380

ctaattggag aaggccaaag gactgtacta cttttgtggg tgtagcagtc gcccagtggg 25440

aaagcgccaa ctaggttaca attgtggtgg ggacaaatta ggggaaatta aattggctta 25500

tagct 25505

<210> 2

<211> 25505

<212> RNA

<213> Porcine Deltacorona Virus ssRNA +

<400> 2

gcaccugaaa ccaagacgcg ucaguaguag ggacuacugg ugcagccaug auagauuggu 60

gucgguagag aacuagcgaa gcuagggaga uaaaauuaua gacuaaugcu auaauuuuua 120

ucuuuagucu auaauuuuau cucccuagcu ucgcuaguuc ucuaccgaca ccaauccagg 180

ugcgucugcc accaaguugg cuacccuucc uaggggcgcu uucgcgcuug cucaccauua 240

gauuaccugg aaaccagcca uucagguugg aguuucccca ggcucuuuug ugugggcauu 300

agcggcuugu gguuuuugca caaaaucuaa gcuacuuacc guuccucuga ccauccacca 360

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ucagaaccuu gaacguuaca ucucacuugc aauugaugca uauccccuca ccaagguuga 14100

cccuauuaag gguaaagucu uuuauuuguu acuagacuac auacguguuc uugyucagga 14160

guuacaggac gguauccuug augcuuucca aucacucacu gacaugucgu auguaaauaa 14220

cuuuaugaau gaggccuuuu augcucagau guaugagcaa aguccuacac uacaggccag 14280

cgguguuugu guggugugua auucacccac uauacugcgc uguggugauu gcauucgucg 14340

accacuacuu uguugcgucu gugccuacca gcauguuacg cagacuacac auaaacguau 14400

cauugcuauc aacaacuaca ucuguagugu ugagaauugc aaugaggaca auguugaaaa 14460

acuuuucauu ucuggcacug cgauuuauug ugagaaucac aaacccacgc ugugcauacc 14520

cauuguagcu aaugguucug uuuuugguau cuaucgccac acugcccgug guagugauga 14580

cauagaccuc uuuaacgagc uugcuacauc uaacuaugac acuauugaac cuuaucagaa 14640

ggccaaucgu gcaccuuuau cacuuaugcu cuucgcugcu gaaaccauua aggcacucga 14700

ggagucuauc aagaagucau augcuaccgc aaccgucaag gauguguaug accaacgcuu 14760

cauuaaacuu cuaugggaac aggguaaaaa gccgccaccc auaacgaaga accacauuuu 14820

cacuggcuac cauuuuaaca agaauggaaa aacccaaguu ggugauuaca uucuugcuaa 14880

aacagauggc agugacacuu auacuuacag aggaacaucu accuacaaac uccaaacagg 14940

ugauguucua gucuuaaugg cacauguugu uacaccgcuc ucagcacccc cuguguuaac 15000

gcagacaaca uaugucagaa aaucacuuuu acccgacucu guuggugcgu cuuauuaugu 15060

gcaacauuuu aagucauaua augagauagc uaugcagagg guuacaacag uauuaggucc 15120

accaggcaca gguaagucaa ccuuugcuau ugguuuggcu aaguacuuuc ccagugcacg 15180

uauuugcuac acugcgucuu cgcaugcagc aaucgaugca cucugugaaa aagcuuucaa 15240

gacaauaccu guaggccaau gcagucguau cguacccaca cguacaacug uugagugcuu 15300

ucaggaguuu gucguaaaua acacaacugc acaguauauc uucucgacua ucaaugccuu 15360

accugacauu aagugugaca uuguaguugu agaugagguu ucuauguuga ccaauuauga 15420

gcuuuccucu gugaaugcuc guuugguuua caaucacauu guguauguug gugauccuua 15480

ucaguuaccu ucaccuagaa cuaugcuuac gucuggccag cuuucgccag cugacuauaa 15540

cguaguuacu gauauaaugg uacaugcagg agcggauguu augcucgaca ugugcuacag 15600

augcccacgu gaaaucguug agacaguguc uaaacuuguc uacgauaaca aacuaaaagc 15660

ggcgaaaccg aacucaagac aguguuacaa gaccauugug aacuuugguc cuggagacgu 15720

ugcucaugag ggacaaucug ccuacaacga agcacaguug cguuucgcac ucgcauuuag 15780

acaacaaaag cggugggaua acgugacuuu cauaucucca uauaaugcua ugaaugugaa 15840

agcauccuua gcagguuucu cuacucagac cguugacucu ucucaagguu cugaguauga 15900

uuauguuauc uuuugcguga ccacugauuc agcacacgca cuuaacaugg cucguuugaa 15960

cguugcccuu acacgcgcaa agauagguau ccuuguggug uuuaggcagg caaacgaacu 16020

uuacaauagu uugcaguuug aaucuauuga uucacagcuu cagucgagug cugagaaaaa 16080

ccucacacca cuguuuaagc gcugcggcua ugaguauaau ggcguccauc cagcucaugc 16140

uuugaccugg caugauugug gugcagagua ccgcugugag gagccacuug cuaaauuagu 16200

aggaguugcc gauggcacuc uuauaucaua caaaacccua guauccacac uuggguuucu 16260

uccaucacuu aaaauugaug cauaucauaa uauguuccua acacgugacg cgugucgcac 16320

cuauguucag aguuggaucg gcauagaugu ugaagcagca cacgccauaa aaccuaacac 16380

cgggacuaac cugccauugc aaauagguuu uaguaccgga aagaauuuuu cagucacucc 16440

agagggaauu uggguaaacg agcacggauc uugcacugag cccgucccug ccaaaauacc 16500

uccuggagaa caauuucguc accuuaaaaa ggacaugcgc caggcgcguc cuuggaaggu 16560

uguucgacgu gagauugcua cucacauugc ugagguagcu ccucacacug auuauauaug 16620

cuuugucacu ugggcucacc agcuugagcu agcgacaaug cgcuacuuug ucaaacuagg 16680

uauggaagag aaaugcuuuu guggcaggcg ggcuuguuuc acuaauggaa cugaguucgc 16740

uugcaaagca caccauucuc ucaccauucc acaaugugau uauguguaca auccauuccu 16800

caucgacgug gcuacguggg gauucucggg acggcuuucc accaaccaug acgcugugug 16860

cacauaucau gcuaaugccc auguugcauc agcugaugca aucaugacgg uauguuuagc 16920

uauccaugaa cuguucagua cuguugacug gaacuuugaa uuuccaguaa cugcugagca 16980

aucgcaacuu aacaaggccu gucgcuuagu acaggcgaau uacuuaaaua uacuacucac 17040

uacaaccaaa gccacggugg uucacgauau ugguaaccca aaagguaucc cuaucgugcg 17100

caaaccuggu guuaaauauc acuucuauga ucaagcaccc auugucaaac acguucaaaa 17160

acuaaaguac aagccagaga uggaggcccg uuucaccgau gguuugacua uguuuuggaa 17220

uuguaauguu gacacauacc cugcuaacgc ccuugugugc cgcuacgaca cucaucggca 17280

gaagcauuua auuggaccua augguucagc acuauauguu aauaagcaug cuuuucucac 17340

cccugagaug cauacuuaug cuacacauaa acucaacuug gcuccacuca ucuacuacuc 17400

caccacagau uguaguagug aacagccuau uguuguuacc uacagagauu gugucacccg 17460

gugcaauacu ggaaaaacuc ucuguccaaa ucaugcucuu gaauaccaag aguuuaucaa 17520

ugcauacaau cucauggcuc gccauggauu uaauguuuac auaccacgca augucaacgu 17580

uuacaacugu uggcuuacuu ucacuaaucu ccaaaaccuu gaaaacuuag cuuacaacug 17640

uuauuauaag aacugcaaug cucacguuga ugggcagcuu gauguaguua uuaauaauaa 17700

cgcuguauau gcuaaggucg acaauaaucu ugucaaacuu uucgacaacc gcacuaacuu 17760

accugucuca guggccuuug aacauuacac uaacaggcau acccguucac ugccaacuac 17820

acagcuguua ucugguuuag gcguaaccgc caccagaaau uucacugugu gguucgacaa 17880

ugauacaauu uuccaauaca cuauuaaugu aucuacguau acugacaucg acccuaguac 17940

ccauguuguc cucugugaug auagguacgg aacagauugg agucaguuua accaacuucc 18000

uaaugcagua uuccucacca aaacuaaggu gaagaaaaca gaaccguuug uuuguacagc 18060

acugacccua aauggcgucg ccauugacgg ugaagagcua uacaucuaug uacgcuauaa 18120

caaucaacug accacauuug cuacuacuug uacacagggu agaaauguug agcaguuuau 18180

accuaaaaca ccuauggaaa gagacuuccu ugagaugucu caacaguccu ucauccagca 18240

acaucaauug caggaacugg guguugaaca cauuaucuau ggugaugauu ccaguccagu 18300

cauuggcgga acucacacac uuaucucacu aguuaaaaac aaguuugaac aucagcuugu 18360

caaccauguu uacaacccag uccagaacug uguuguuacc ucaccuaacg caagcuccaa 18420

gaacguuugc acuguucuug auguucuucu ugaugacuac auugacauca uaagacaagc 18480

acaugccagu uacacaagua aauccaaagu auucacugug ucaauugaca accaacaaau 18540

uagauucaug cuuuggcaug augagcaagu caagacuugc uacccaaucu uacagucacu 18600

uaccaauggu uaccagaugc caucugugua caaaacauug guuacugacu uacaaccagc 18660

ugacaucccu aauuaucauu ccuacacccc ccgggugccu ggaguaguua agaauguuau 18720

caaguaccgc caacuuuuca acuacauagu uaaaaaggau agguuggcag uaccacacaa 18780

uaugaccgua uuacaccuug gagcugcauc ugcacuaggu acagcaccag guucuucagu 18840

cauaaaacaa auguuuccug aaggaacugu ucuuauugac cucgauauaa gagaguucac 18900

uucagaugcu aaccaaauaa uaguuacaga cuacagaacu uacauaccac cacaccacgu 18960

agacgucaua uuuucugacc ucuacuguug ugaugacaua cacuucuuug acaaucuaau 19020

aaggauaguu aaggagaggc ucgcccucgg ugguucuauc uuuguuaaga uaacugaaca 19080

uucauucuca cccgaacucu acucacuugc ggguugguuc gaugauuauc aacuauuuug 19140

cacagcaguc aaugccucgu cuucagaagc auuuuuaugc uguuuuaauu auuuggggcu 19200

ugcuaaggaa aacauuaaug guuuuaacuu acaugcuucc uauauucaau ggcgcaauga 19260

aauagcguug acaccaaccu auucuccuuu agcggacaac ccggcuacgg ccuguaagcu 19320

aaaagcaacg ccuauuaucu cggcucguga guuagagaag aagccuauuc uucgcuaucu 19380

cguugcauca gggcgccuuc uugugaggcc accagaaugc agagagcucu auugauuaug 19440

accuuauuuu gucucguucg agcaaaguuu gcugaugauc uacucgauuu gcucaccuuc 19500

ccgggugcac aucgcuucuu acauaaaccc acgaggaauu ccagcagucu cuacucgcgg 19560

gcuaauaaua auuuugaugu uggcguucuu ccuggcuacc ccacuaagaa cguuaaccuc 19620

uucucaccac uuacuaacuc cacuuugccc auuaauggcc uucaucggag uuaccaacca 19680

cucaugcuga auugucuuac uaaaauaacu aaccacacuc ucagcaugua ucuccuaccu 19740

agugagauac aaacuuauag cugcggcggu gccaugguua aauaccagac acaugaugca 19800

guucguauca uuuuagaccu cacugccacu gaccacaucu cuguugaagu cguuggccaa 19860

cauggugaaa auuauguguu uguuugcagu gagcagucua ccuacaccac ugcauuacac 19920

aaaucuaccu ucuucucacu uaauucugag cuuuauugcu uuacuaauaa caccuacuua 19980

gguauucuuc caccugauuu aacugacuuu acggucuacc guacugguca guucuaugcu 20040

aaugguuacc uuuuagguac uuuaccuauu acgguuaacu auguuagguu guaucggggu 20100

cauuugucug ccaauagugc ccacuuugcc cuugcaaacc uaaccgauac acucauaaca 20160

cuuaccaaua cuacuauauc gcaaaucacu uauugugaua agucuguagu ugauucaaua 20220

gcaugccagc gcucuucuca cgaaguggag gauggguuuu acuccaaccc uaaaucugcc 20280

guuagagcua ggcaacguac uauuguuaca cuaccuaagc ucccugagcu ugaaguagug 20340

caguuaaaua uuucugcaca cauggauuuu ggcgaagcca gacuugacag cguuaccauc 20400

aaugguaaca cauccuauug ugucacuaag ccuuacuuca ggcuugaaac uaacuuuaug 20460

uguacagguu gcacuaugaa ucugcgcacu gauaccugua guuuugaccu gucagcagua 20520

aacaauggca ugucauucuc ucaauucugu cuaagcacug aaucuggugc uugugagaug 20580

aaaauuauug uuaccuacgu auggaauuac uugcuaaggc agcguuugua uguuacugcu 20640

guagagggcc agacucacac uggaaccacu ucaguacaug caacagacac uucuagugua 20700

aucacugaug ucugcacuga cuacacuauc uauggagucu cugguacugg cauuauuaag 20760

ccaucagauc ucuuauugca caauggcaua guauucaccu cuccaacagg ugagcuuuau 20820

gcauuuaaaa auauaaccac uggcaaaacc cuucaggucu uaccguguga aaccccuucu 20880

caacugauug ugauaaacaa caccguuguc ggugcuauca cauccaguaa uucaacugaa 20940

aauaauaggu uuacuacuac uauugucaca ccuacuuucu uuuauuccac aaaugccacc 21000

acuuucaacu gcacuaagcc uguuuugucc uauggaccua ucagcgugug uagugauggu 21060

gcaauugugg gaacauccac auuacagaau acucgaccau ccauaguuuc acuauacgau 21120

ggcgaaguug aaauaccauc ugcauuuucu cuuuccguuc agacggagua cuugcaaguu 21180

caagcagagc aaguuauagu ugauuguccu caguauguau gcaauggcaa cagccguugu 21240

cuacaauuac uggcacaaua caccucagcu ugcucuaaga uugaagcagc ucugcauucc 21300

ucugcacagu uggauagcag agagauuaua aauauguuuc aaacaucaac acaguccuug 21360

caguuagcua auauuaccaa cuucaagggu gacuacaauu uuagcagcau acuaaccacc 21420

agaauuggug gcagaucugc uauugaagac cuucuuuuua auaaaguugu uacuaguggc 21480

cuuggcacug uugaucagga cuacaaaucc ugcucuagag acauggccau cgcugacuua 21540

guuuguuccc aguauuacaa uggcaucaug guucuaccug guguuguuga ugcugagaaa 21600

auggcaaugu auacuggcuc ucuuacugga gcuaugguau uuggaggacu gacugccgca 21660

gcggcaauac cauuugccac ggcaguacaa gcucgccuca auuaugucgc acugcaaaca 21720

aauguacuac aagaaaacca gaaaauucuu gcagaaucau uuaaccaagc aguuggcaau 21780

auaucacuug cacuaucuuc uguuaaugau gccauccagc aaacuucuga ggcucuuaac 21840

accguagcua uugcuauuaa aaagauucaa acaguuguua accagcaggg cgaggcauua 21900

ucacaccuga cugcacagcu gucaaacaau uuucaagcaa uuucgacuuc uauucaagac 21960

auuuacaacc gucuugagga aguagaggcu aaccagcaag uugaccgucu caucacagga 22020

cgguuggcug cacuuaaugc auauguuacu caguuacuca aucagauguc ucagauuaga 22080

caaucucgau uguuagcuca gcaaaagauu aaugagugug ucaaaucaca gucguccaga 22140

uacgguuucu guggaaaugg cacacacauc uucucacuua cacagacugc accaaauggc 22200

auauuuuuca ugcaugcagu gcuaguaccc aacaaauuca cacgugucaa cgcuucugcc 22260

ggcauuugug uggauaauac gagaggcuac ucauugcagc cucaacuuau acucuaccag 22320

uuuaauaacu ccuggagagu uacaccuaga aauauguaug aacccagacu gccccggcag 22380

gcugauuuca uacaauuaac ugauugcagc guuacuuuuu acaacaccac cgcugcuaau 22440

cuucccaaua uuaucccuga cauuauagau gucaaucaaa cagucaguga uauuauugac 22500

aauuuaccua cagcaacacc uccucagugg gauguuggua ucuauaacaa cacuauucuc 22560

aaccucaccg uugagauuaa ugaucuacaa gagcggucua aaaaccucuc acagauugca 22620

gaucguuuac aaaauuauau ugacaaucuu aacaauacuc uaguugaccu ugaauggcuc 22680

aacagagugg aaacuuaccu uaaauggccg ugguauauau ggcuugccau ugcccuggcu 22740

cuuauugcau uugugacaau ccucauaaca aucuuucuuu guacugguug uugugguggu 22800

ugcuuugguu guuguggcgg uuguuuuggc cuuuucucua agaagaaaag guauaccgac 22860

gaccaaccaa caccguccuu uaaguuuaag gaaugguagu cgacgacugg gccguuacca 22920

ucccuggaca auauauuauu gcuauacuag uugucaucug cauuggugug gcacuacuuu 22980

uuauuaauac uugcuuagcu uguguuaaau uauuuuacaa gugcuaccua ggggcagcau 23040

aucuuguuag gccuauuaua guguacuacu ccaagccgaa ccccguaccu gaggaugagu 23100

uuguaaaagu acaccaauuu ccuagaaaca cucacuaugu cugacgcaga agaguggcaa 23160

auuauuguuu ucauugcgau cauaugggcg cuuggcguca uccuccaggg aggcuaugcc 23220

acgcguaauc gugugaucua uguuauuaaa cuuauucugc uuuggcugcu ccaacccuuc 23280

acccuagugg ugaccauuug gaccgcaguc gacagaucau cuaagaagga cgcaguuuuc 23340

auugugucca uaauuuuugc cguacugacc uucauauccu gggccaagua cugguaugac 23400

ucaauucguu uauuaaugaa aaccagaucu gcaugggcac ucucaccuga gaguagacuc 23460

cuugcaggga uuauggaucc aauggguaca uggaggugca uucccauuga ccacauggcu 23520

ccaauucuca caccagucgu uaagcauggc aagcucaagc uacaugggca agagcuggcc 23580

aauggcauau cagucagaaa uccgccacag gauaugguga uagugucacc aagugacacc 23640

uuucacuaca cuuuuaagaa accuguggaa ucaaacaacg auccagaauu ugcuguucug 23700

auauaccagg gugaccgcgc uucaaacgcu ggacuucaca ccauaaccac uucaaaggcc 23760

ggugacgcuc gccuguauaa guauauguaa ugugcaacug ccaucugcag cugcgagauu 23820

uauauagauu gugcaauaag cggcacauca gaagagagga uguuccugag cuuauugacc 23880

cucucguuaa aacucgcugu uuugcuuaca gucucguggu ucuugcuaau gcuaauccaa 23940

uugcauuuag cauacuaccu cggaaaauuc uuaucaaugg ugagccuuua cugcuugaau 24000

augguagcau auaugguaaa gacuuuauca uucgaccauc gcuccaaguc auucuugaag 24060

augaauuaaa uuaaaguuuu gacaccaauc uaucauggcu gcaccaguag ucccuacuac 24120

ugacgcgucu ugguuucagg ugcucaaagc ucaaaacaaa aaggcuacuc auccucaguu 24180

ucguggcaau ggaguuccgc uuaacuccgc caucaaaccc guugaaaacc auggcuacug 24240

gcugcguuac accagacaaa agccaggugg uacucccauu ccuccauccu augccuuuua 24300

uuauacuggc acagguccca gaggaaaucu uaaguauggu gaacucccuc cuaaugauac 24360

cccagcaacc acucguguua cuuggguuaa ggguucggga gcugacacuu cuauuaaacc 24420

ucauguugcc aaacgcaacc ccaacaaucc uaaacaucag cugcuaccuc uccgauuccc 24480

aaccggagau ggcccagcuc aagguuucag aguugacccc uucaacgcua gaggaagacc 24540

ucaggagcgu ggaaguggcc caagaucuca aucuguuaac uccagaggca caggcaauca 24600

gcccaggaaa cgcgaccaau cugcaccagc ugcgguacgu cguaagaccc agcaucaagc 24660

ucccaagcgg acuuuaccca aggguaaaac cauuucucag guauuuggca accggucucg 24720

uacuggugcc aaugucggcu cugcagacac ugagaagacg gguauggcug auccucgcau 24780

cauggcucua gccagacaug ugccuggugu ucaggaaaug cuuuuugcug gccaccuuga 24840

gagcaacuuu caggcggggg caauuacccu uaccuucucc uacucaauca cagucaagga 24900

ggguucuccu gacuaugaga gacuuaagga ugcgcucaau acggucguua accagaccua 24960

ugagccaccu accaaaccaa cuaaggacaa gaagccugac aaacaagacc agucugcuaa 25020

acccaaacag cagaagaaac cuaaaaaggu aacucugcca gcagacaaac aggauuggga 25080

gugggaugau gcuuuugaga uaaagcagga aucagcagcg uagacaucaa ucuaugucug 25140

uuaaacccac ccaacuccac ucaaauaucu cuuugguucc agagagucgu aguguauagc 25200

cagagagcca gucagagggc gcuaucaugc aaacuagggc uggcuacucu agcacagaau 25260

cacaucccga uaaucaacag ugcuagaagg uugauuauac cauuuaauau gccgaggcca 25320

cgcggaguac gaucgagggu acagcauaau cucaacuuuu guugagccac aauuuuaauc 25380

cuaauuggag aaggccaaag gacuguacua cuuuuguggg uguagcaguc gcccaguggg 25440

aaagcgccaa cuagguuaca auuguggugg ggacaaauua ggggaaauua aauuggcuua 25500

uagcu 25505

<210> 3

<211> 3459

<212> DNA

<213> Artificial Sequence

<220>

<223> Deltacorona Virus SPIKE antigen CDS (NSVL) cDNA

<400> 3

atgaccttat tttgtctcgt tcgagcaaag tttgctgatg atctactcga tttgctcacc 60

ttcccgggtg cacatcgctt cttacataaa cccacgagga attccagcag tctctactcg 120

cgggctaata ataattttga tgttggcgtt cttcctggct accccactaa gaacgttaac 180

ctcttctcac cacttactaa ctccactttg cccattaatg gccttcatcg gagttaccaa 240

ccactcatgc tgaattgtct tactaaaata actaaccaca ctctcagcat gtatctccta 300

cctagtgaga tacaaactta tagctgcggc ggtgccatgg ttaaatacca gacacatgat 360

gcagttcgta tcattttaga cctcactgcc actgaccaca tctctgttga agtcgttggc 420

caacatggtg aaaattatgt gtttgtttgc agtgagcagt ctacctacac cactgcatta 480

cacaaatcta ccttcttctc acttaattct gagctttatt gctttactaa taacacctac 540

ttaggtattc ttccacctga tttaactgac tttacggtct accgtactgg tcagttctat 600

gctaatggtt accttttagg tactttacct attacggtta actatgttag gttgtatcgg 660

ggtcatttgt ctgccaatag tgcccacttt gcccttgcaa acctaaccga tacactcata 720

acacttacca atactactat atcgcaaatc acttattgtg ataagtctgt agttgattca 780

atagcatgcc agcgctcttc tcacgaagtg gaggatgggt tttactccaa ccctaaatct 840

gccgttagag ctaggcaacg tactattgtt acactaccta agctccctga gcttgaagta 900

gtgcagttaa atatttctgc acacatggat tttggcgaag ccagacttga cagcgttacc 960

atcaatggta acacatccta ttgtgtcact aagccttact tcaggcttga aactaacttt 1020

atgtgtacag gttgcactat gaatctgcgc actgatacct gtagttttga cctgtcagca 1080

gtaaacaatg gcatgtcatt ctctcaattc tgtctaagca ctgaatctgg tgcttgtgag 1140

atgaaaatta ttgttaccta cgtatggaat tacttgctaa ggcagcgttt gtatgttact 1200

gctgtagagg gccagactca cactggaacc acttcagtac atgcaacaga cacttctagt 1260

gtaatcactg atgtctgcac tgactacact atctatggag tctctggtac tggcattatt 1320

aagccatcag atctcttatt gcacaatggc atagtattca cctctccaac aggtgagctt 1380

tatgcattta aaaatataac cactggcaaa acccttcagg tcttaccgtg tgaaacccct 1440

tctcaactga ttgtgataaa caacaccgtt gtcggtgcta tcacatccag taattcaact 1500

gaaaataata ggtttactac tactattgtc acacctactt tcttttattc cacaaatgcc 1560

accactttca actgcactaa gcctgttttg tcctatggac ctatcagcgt gtgtagtgat 1620

ggtgcaattg tgggaacatc cacattacag aatactcgac catccatagt ttcactatac 1680

gatggcgaag ttgaaatacc atctgcattt tctctttccg ttcagacgga gtacttgcaa 1740

gttcaagcag agcaagttat agttgattgt cctcagtatg tatgcaatgg caacagccgt 1800

tgtctacaat tactggcaca atacacctca gcttgctcta agattgaagc agctctgcat 1860

tcctctgcac agttggatag cagagagatt ataaatatgt ttcaaacatc aacacagtcc 1920

ttgcagttag ctaatattac caacttcaag ggtgactaca attttagcag catactaacc 1980

accagaattg gtggcagatc tgctattgaa gaccttcttt ttaataaagt tgttactagt 2040

ggccttggca ctgttgatca ggactacaaa tcctgctcta gagacatggc catcgctgac 2100

ttagtttgtt cccagtatta caatggcatc atggttctac ctggtgttgt tgatgctgag 2160

aaaatggcaa tgtatactgg ctctcttact ggagctatgg tatttggagg actgactgcc 2220

gcagcggcaa taccatttgc cacggcagta caagctcgcc tcaattatgt cgcactgcaa 2280

acaaatgtac tacaagaaaa ccagaaaatt cttgcagaat catttaacca agcagttggc 2340

aatatatcac ttgcactatc ttctgttaat gatgccatcc agcaaacttc tgaggctctt 2400

aacaccgtag ctattgctat taaaaagatt caaacagttg ttaaccagca gggcgaggca 2460

ttatcacacc tgactgcaca gctgtcaaac aattttcaag caatttcgac ttctattcaa 2520

gacatttaca accgtcttga ggaagtagag gctaaccagc aagttgaccg tctcatcaca 2580

ggacggttgg ctgcacttaa tgcatatgtt actcagttac tcaatcagat gtctcagatt 2640

agacaatctc gattgttagc tcagcaaaag attaatgagt gtgtcaaatc acagtcgtcc 2700

agatacggtt tctgtggaaa tggcacacac atcttctcac ttacacagac tgcaccaaat 2760

ggcatatttt tcatgcatgc agtgctagta cccaacaaat tcacacgtgt caacgcttct 2820

gccggcattt gtgtggataa tacgagaggc tactcattgc agcctcaact tatactctac 2880

cagtttaata actcctggag agttacacct agaaatatgt atgaacccag actgccccgg 2940

caggctgatt tcatacaatt aactgattgc agcgttactt tttacaacac caccgctgct 3000

aatcttccca atattatccc tgacattata gatgtcaatc aaacagtcag tgatattatt 3060

gacaatttac ctacagcaac acctcctcag tgggatgttg gtatctataa caacactatt 3120

ctcaacctca ccgttgagat taatgatcta caagagcggt ctaaaaacct ctcacagatt 3180

gcagatcgtt tacaaaatta tattgacaat cttaacaata ctctagttga ccttgaatgg 3240

ctcaacagag tggaaactta ccttaaatgg ccgtggtata tatggcttgc cattgccctg 3300

gctcttattg catttgtgac aatcctcata acaatctttc tttgtactgg ttgttgtggt 3360

ggttgctttg gttgttgtgg cggttgtttt ggccttttct ctaagaagaa aaggtatacc 3420

gacgaccaac caacaccgtc ctttaagttt aaggaatgg 3459

<210> 4

<211> 1153

<212> PRT

<213> Porcine Deltacorona Virus

<400> 4

Met Thr Leu Phe Cys Leu Val Arg Ala Lys Phe Ala Asp Asp Leu Leu

1 5 10 15

Asp Leu Leu Thr Phe Pro Gly Ala His Arg Phe Leu His Lys Pro Thr

20 25 30

Arg Asn Ser Ser Ser Leu Tyr Ser Arg Ala Asn Asn Asn Phe Asp Val

35 40 45

Gly Val Leu Pro Gly Tyr Pro Thr Lys Asn Val Asn Leu Phe Ser Pro

50 55 60

Leu Thr Asn Ser Thr Leu Pro Ile Asn Gly Leu His Arg Ser Tyr Gln

65 70 75 80

Pro Leu Met Leu Asn Cys Leu Thr Lys Ile Thr Asn His Thr Leu Ser

85 90 95

Met Tyr Leu Leu Pro Ser Glu Ile Gln Thr Tyr Ser Cys Gly Gly Ala

100 105 110

Met Val Lys Tyr Gln Thr His Asp Ala Val Arg Ile Ile Leu Asp Leu

115 120 125

Thr Ala Thr Asp His Ile Ser Val Glu Val Val Gly Gln His Gly Glu

130 135 140

Asn Tyr Val Phe Val Cys Ser Glu Gln Ser Thr Tyr Thr Thr Ala Leu

145 150 155 160

His Lys Ser Thr Phe Phe Ser Leu Asn Ser Glu Leu Tyr Cys Phe Thr

165 170 175

Asn Asn Thr Tyr Leu Gly Ile Leu Pro Pro Asp Leu Thr Asp Phe Thr

180 185 190

Val Tyr Arg Thr Gly Gln Phe Tyr Ala Asn Gly Tyr Leu Leu Gly Thr

195 200 205

Leu Pro Ile Thr Val Asn Tyr Val Arg Leu Tyr Arg Gly His Leu Ser

210 215 220

Ala Asn Ser Ala His Phe Ala Leu Ala Asn Leu Thr Asp Thr Leu Ile

225 230 235 240

Thr Leu Thr Asn Thr Thr Ile Ser Gln Ile Thr Tyr Cys Asp Lys Ser

245 250 255

Val Val Asp Ser Ile Ala Cys Gln Arg Ser Ser His Glu Val Glu Asp

260 265 270

Gly Phe Tyr Ser Asn Pro Lys Ser Ala Val Arg Ala Arg Gln Arg Thr

275 280 285

Ile Val Thr Leu Pro Lys Leu Pro Glu Leu Glu Val Val Gln Leu Asn

290 295 300

Ile Ser Ala His Met Asp Phe Gly Glu Ala Arg Leu Asp Ser Val Thr

305 310 315 320

Ile Asn Gly Asn Thr Ser Tyr Cys Val Thr Lys Pro Tyr Phe Arg Leu

325 330 335

Glu Thr Asn Phe Met Cys Thr Gly Cys Thr Met Asn Leu Arg Thr Asp

340 345 350

Thr Cys Ser Phe Asp Leu Ser Ala Val Asn Asn Gly Met Ser Phe Ser

355 360 365

Gln Phe Cys Leu Ser Thr Glu Ser Gly Ala Cys Glu Met Lys Ile Ile

370 375 380

Val Thr Tyr Val Trp Asn Tyr Leu Leu Arg Gln Arg Leu Tyr Val Thr

385 390 395 400

Ala Val Glu Gly Gln Thr His Thr Gly Thr Thr Ser Val His Ala Thr

405 410 415

Asp Thr Ser Ser Val Ile Thr Asp Val Cys Thr Asp Tyr Thr Ile Tyr

420 425 430

Gly Val Ser Gly Thr Gly Ile Ile Lys Pro Ser Asp Leu Leu Leu His

435 440 445

Asn Gly Ile Val Phe Thr Ser Pro Thr Gly Glu Leu Tyr Ala Phe Lys

450 455 460

Asn Ile Thr Thr Gly Lys Thr Leu Gln Val Leu Pro Cys Glu Thr Pro

465 470 475 480

Ser Gln Leu Ile Val Ile Asn Asn Thr Val Val Gly Ala Ile Thr Ser

485 490 495

Ser Asn Ser Thr Glu Asn Asn Arg Phe Thr Thr Thr Ile Val Thr Pro

500 505 510

Thr Phe Phe Tyr Ser Thr Asn Ala Thr Thr Phe Asn Cys Thr Lys Pro

515 520 525

Val Leu Ser Tyr Gly Pro Ile Ser Val Cys Ser Asp Gly Ala Ile Val

530 535 540

Gly Thr Ser Thr Leu Gln Asn Thr Arg Pro Ser Ile Val Ser Leu Tyr

545 550 555 560

Asp Gly Glu Val Glu Ile Pro Ser Ala Phe Ser Leu Ser Val Gln Thr

565 570 575

Glu Tyr Leu Gln Val Gln Ala Glu Gln Val Ile Val Asp Cys Pro Gln

580 585 590

Tyr Val Cys Asn Gly Asn Ser Arg Cys Leu Gln Leu Leu Ala Gln Tyr

595 600 605

Thr Ser Ala Cys Ser Lys Ile Glu Ala Ala Leu His Ser Ser Ala Gln

610 615 620

Leu Asp Ser Arg Glu Ile Ile Asn Met Phe Gln Thr Ser Thr Gln Ser

625 630 635 640

Leu Gln Leu Ala Asn Ile Thr Asn Phe Lys Gly Asp Tyr Asn Phe Ser

645 650 655

Ser Ile Leu Thr Thr Arg Ile Gly Gly Arg Ser Ala Ile Glu Asp Leu

660 665 670

Leu Phe Asn Lys Val Val Thr Ser Gly Leu Gly Thr Val Asp Gln Asp

675 680 685

Tyr Lys Ser Cys Ser Arg Asp Met Ala Ile Ala Asp Leu Val Cys Ser

690 695 700

Gln Tyr Tyr Asn Gly Ile Met Val Leu Pro Gly Val Val Asp Ala Glu

705 710 715 720

Lys Met Ala Met Tyr Thr Gly Ser Leu Thr Gly Ala Met Val Phe Gly

725 730 735

Gly Leu Thr Ala Ala Ala Ala Ile Pro Phe Ala Thr Ala Val Gln Ala

740 745 750

Arg Leu Asn Tyr Val Ala Leu Gln Thr Asn Val Leu Gln Glu Asn Gln

755 760 765

Lys Ile Leu Ala Glu Ser Phe Asn Gln Ala Val Gly Asn Ile Ser Leu

770 775 780

Ala Leu Ser Ser Val Asn Asp Ala Ile Gln Gln Thr Ser Glu Ala Leu

785 790 795 800

Asn Thr Val Ala Ile Ala Ile Lys Lys Ile Gln Thr Val Val Asn Gln

805 810 815

Gln Gly Glu Ala Leu Ser His Leu Thr Ala Gln Leu Ser Asn Asn Phe

820 825 830

Gln Ala Ile Ser Thr Ser Ile Gln Asp Ile Tyr Asn Arg Leu Glu Glu

835 840 845

Val Glu Ala Asn Gln Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ala

850 855 860

Ala Leu Asn Ala Tyr Val Thr Gln Leu Leu Asn Gln Met Ser Gln Ile

865 870 875 880

Arg Gln Ser Arg Leu Leu Ala Gln Gln Lys Ile Asn Glu Cys Val Lys

885 890 895

Ser Gln Ser Ser Arg Tyr Gly Phe Cys Gly Asn Gly Thr His Ile Phe

900 905 910

Ser Leu Thr Gln Thr Ala Pro Asn Gly Ile Phe Phe Met His Ala Val

915 920 925

Leu Val Pro Asn Lys Phe Thr Arg Val Asn Ala Ser Ala Gly Ile Cys

930 935 940

Val Asp Asn Thr Arg Gly Tyr Ser Leu Gln Pro Gln Leu Ile Leu Tyr

945 950 955 960

Gln Phe Asn Asn Ser Trp Arg Val Thr Pro Arg Asn Met Tyr Glu Pro

965 970 975

Arg Leu Pro Arg Gln Ala Asp Phe Ile Gln Leu Thr Asp Cys Ser Val

980 985 990

Thr Phe Tyr Asn Thr Thr Ala Ala Asn Leu Pro Asn Ile Ile Pro Asp

995 1000 1005

Ile Ile Asp Val Asn Gln Thr Val Ser Asp Ile Ile Asp Asn Leu

1010 1015 1020

Pro Thr Ala Thr Pro Pro Gln Trp Asp Val Gly Ile Tyr Asn Asn

1025 1030 1035

Thr Ile Leu Asn Leu Thr Val Glu Ile Asn Asp Leu Gln Glu Arg

1040 1045 1050

Ser Lys Asn Leu Ser Gln Ile Ala Asp Arg Leu Gln Asn Tyr Ile

1055 1060 1065

Asp Asn Leu Asn Asn Thr Leu Val Asp Leu Glu Trp Leu Asn Arg

1070 1075 1080

Val Glu Thr Tyr Leu Lys Trp Pro Trp Tyr Ile Trp Leu Ala Ile

1085 1090 1095

Ala Leu Ala Leu Ile Ala Phe Val Thr Ile Leu Ile Thr Ile Phe

1100 1105 1110

Leu Cys Thr Gly Cys Cys Gly Gly Cys Phe Gly Cys Cys Gly Gly

1115 1120 1125

Cys Phe Gly Leu Phe Ser Lys Lys Lys Arg Tyr Thr Asp Asp Gln

1130 1135 1140

Pro Thr Pro Ser Phe Lys Phe Lys Glu Trp

1145 1150

<210> 5

<211> 25426

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus BIVI-Isolate 2.0307 Genome cDNA

<400> 5

gattggtgtc ggtagagaac tagcgaagct agggagataa aattatagac taatgctata 60

atttttatct ttagtctata attttatctc cctagcttcg ctagttctct accgacacca 120

atccaggtgc gtctgccacc aagttggcta cccttcctag gggcgctttc gcgcttgctc 180

accattagat tacctggaaa ccagccattc aggttggagt ttccccaggc tcttttgtgt 240

gggcattagc ggcttgtggt ttttgcacaa aatctaagct acttaccgtt cctctgacca 300

tccaccactt ctatagacag cactgattac cgtagggttt aagtcacacc ggtctgcacc 360

gcccgtcagc ggacacatta cccagcatag cactccttgc accgagccta ggtaggataa 420

aaccccctac cgggtgactc ttaaggcgtt tcctccacgg gatagccact agtcactagg 480

tgtaagtgat ctgatctggg cgtattgtgt tgcgcaagtg tgatacccat aggagcgtgg 540

aatcctattc tgcggctcag tgcctgatat agctgtgaaa tggccaagaa caagtccaag 600

cgcgacgcta ttgcgttgcc tgaaaatgta ccaccacctc tgcaactttt cattcatgtt 660

gcagctgctg aagagggtca ccctaaggtt actacttacc ttggcaacta taacctctat 720

gccaccaagg ctccgcctgg cgtgcaggtt cttagtgcta aaacctctct tactgacttt 780

gagaatgtct ttggagctca acccaccttg cgatcaattc gtaatctggt ttgtgaggct 840

cgctcggctg aatggacaac ttccaagaat gcttttgcac tcaaagccac tcaacttgac 900

tactctgatg ccgttttgag ggcaatgatt cgtttctgcc ctccaaaggt gtccacactc 960

gctgcctttg ctctttttgg ccgattggtt aaaattgagg acaaggaact tgctgagtta 1020

gctcgtgata ctgcccttga gttggcgtac acggctaaaa ttggtacatc tcttgctgac 1080

acgagatctg tctcacttat tcataaggac gcttatctaa ctctcagtaa tgaggttgtt 1140

ggcgtaactt ttactgccgc acttatggca aaggctacca ctgttaatgg agcaatgcaa 1200

tactcaaact tttacctcta ccctcgtgcc actattaagg taaccgatgg taaggctgaa 1260

gcaattgcaa ctaagcctct gtctgctgcc actaaaggca agcaaatcac agaggatgtc 1320

aacctcctcc ctgactatca gcagctgctt gttgatcaag tgactggcac tgaggttaag 1380

gttggagctc taacctatgt taagaccact gattcgccac ccctttactt tcccaaagtc 1440

aagggtggtg ttattggtat tgcacttaag cagcagggca ctgcggctaa gaagctcaat 1500

gtagtcttcc atgctcaacc tgatgatgtt ctgctagcct tcatacaact tcagcaattc 1560

ttgaaccgta cttcggattc aagtgttgaa attactgatt gccagagtta tgaagtatct 1620

ccaactgtga cggtcaaaat tggcccgtct aaacctgggg atgtcatcgt ggctactgat 1680

gaggaatacc ttaaatgctt tgaaacccct gaggtaggta ggctctataa ggttttccaa 1740

actcaatctt gggctatcat tgagcgtgcc ttctccagtt tgaagatccg cgtgtccaaa 1800

gctttatcag catttataag ttttctgcaa aaccttgcag ataactttac tgcaataagt 1860

ggtgttgtca ctgcactcat tcgtgaactc caggatctta ccctggatgt ggcgacacgt 1920

atcactaaca tacaatttgt ttaccgtgcc ggtaagctta ttgtcgacac gacaagtgtc 1980

atagctaaac ttttccagcc attttgtgat tttatatcac ctttccttcg gaaagttgct 2040

ggttttgcaa tttacactgt tggtaatcgc atgcttatgt ttaccagcac tggcaccttt 2100

cttctcacaa aggcaactac taagatactc aataaggcaa agtacatctt tgatgtggag 2160

cctgagtacc cagtagatgt aacaacatcc aaagttgtag tacatgaagc actccagcaa 2220

accgacacta agcctactag agctctggag gctgttgatg tcgttgttgg taatactgta 2280

ctgcaaatgg ctactgatgg cactgcgttc tacccatcgg atggtacgca cgcctctctt 2340

ccaggattca aagcaggttc ggatgagctt ttcataagct tcaactgcga cctctttgat 2400

gatgagacta atgctcaaat caacgaaaca ctcgctgcat atgagcttaa ccaactagtg 2460

gctccaggtg attctacacc gcgtcaaatt gcgacgttgg ttgtcgatac acttgcagat 2520

gctataacag accactttcc ggagaaaacc attgatctac ctgaagacta tcaagtcttt 2580

tctgaccatg atgacctccc actcgcacaa taccacatcc ctgatcacct gagcctgtat 2640

attcaggcta tggaaggtga agatgatagt ggtgatgaaa tatgtattga ggacgatgat 2700

tacgactgtc ctcaagccga cgaagacaca gaaggagtaa ttccccaaca gtgggaactt 2760

cctgatgttg ataaattttt actcaagatc caggaacgga agaccagcag cgacgaagta 2820

cttagcgtcg acgtctatcc taaaccagag ccggtcggca atgttgggat tgacgacagc 2880

gcgtcggaaa agaagccaaa tggggactca gtaccggatc ctgaggtcca tccaacacta 2940

gagagtgtgg atgttgaacg accaaccgaa acagcaaacc aggctgttga agacaaacct 3000

tctgatacca cctttgtggt tgatgaggaa caattacaag aatcaacacc agaacatgaa 3060

ctccgctcct atgaagggga gtttgattct gatgatgaaa ttattattcc tatagtacca 3120

gtaacacctg cggatttaaa accacagact attactataa aggagtactt taagtctgaa 3180

aaacttgaga ctattaacga aggatccaca gagtcagtta cacaatctga cgattcgttt 3240

gacgagtcat ttgttgatgc tgagtctgat gatccacaag atcctgctgt atatgatgat 3300

acaacaatta taacggacag cactgatgta ggcgatgagc ctgagacaac tctagctacc 3360

atcgttaaca cacctctgac actcgataat aacttgccac ctgaagccat taaacaaccc 3420

agcccaacta aggttgagtt agttgttggt gaattggcga gtattaaatt tgacaattct 3480

gttctagtca accctgctaa tgcgcaatta acaaatggcg gtggagctgc tcgtgcaatt 3540

gcaaaattag ctggtccaaa atatcaagag tactgtaata gtgtggctcc tatctcagga 3600

ccgcttacca cggactcttt tgatgccaag aaatttggtg tagcctgcat cttgcatgta 3660

gtgccaccca aaggttctga ccctaatgta caagaactcc tgtatcaagc ttacaagagt 3720

atccttactg aaccagcaca ctatgttata cctatactag gtgctggtat ctttggatgc 3780

aatccagtcc actctctgga tgcgttcagg aaagcatgtc caagtgacat aggtcgtgtc 3840

acccttgtca ctatgaacaa aaaccatttg caggtgtggg atgctctcaa taggaccatt 3900

gtacgcacca ctactgacta tgatcaagtt accaccaagg cccttacacc ccagggagtg 3960

ttagaagcca atctctttga tggtgaggac tttgttcaag aaccaaaacc cggtcaaatc 4020

taccttgagg ttactgaaga agttcagaac caagccaagg aacttgacct taaccttcag 4080

caatactgcg tctacctgaa gacttgccac cataaatggg ttgtgagtcg tacgaacggg 4140

ttgatgcatc taaaacaaaa agataacaat tgttttgtta gtgcaggtgt aaacctgttt 4200

caaaacactg cttatcaact tagacctgct attgatgctc tctataggga gtatcttaat 4260

ggtaatccaa atagatttgt tgcttggatc tacgcatcca ctaaccgtcg tgttggtgag 4320

atgggttgtc cacagcaagt tatttctttg ctcgttagta actctgacgc agcattttca 4380

gcaactacag cctgttgtaa cacctacttt aaccacacag gtgttatttc agtagctcgt 4440

gaatatgacc caatacaacc aaaggtctac tgcatgaagt gtgatgtgtg gactcccttt 4500

acaccccaga gtggaaaagg tgcagttgca attggtattt ctgcagatga acctaccggt 4560

cctgccatta aatttgccgc agctcactgc tggtacacta atggcaagaa aacagttaat 4620

ggctatgaca ctaaagctaa tgttgtagct acctatcata ggtttgacgt gcctaagcct 4680

caacttgtcg aggacgtggt tgcgctgcct actaaaaatg actttgaagt tctcaatgtt 4740

gaagaactgc cgcaggatag tgtgctccat ttggacccac ctcctgtaca ggccttacaa 4800

cctaaggcta accaacacat tgagattcta gaaaacccag attatctgga cattttggat 4860

ctttggattc gtaaacccaa attcatcctc gtaaagtcgt ggagtgtttt gggtagagca 4920

ctatgtaagg caggtaaagt tgtctttgtc agtgcttcgc ttttgacccg tttctacaat 4980

taccttgtag agattggtgc tcttgactca acaataaggt tgtcagtcga tcttacctgt 5040

aaatttgtta gaacggttct cccatcgtct aacactgtac acaaaacttg tcttggtctg 5100

tattattcag cccagacact ttttgtttct ttagcaccat tccttatgtt accagctgta 5160

gttagtctgc ttaattcagg ctatacaatt ggcacatatt tgtatgcaaa aactggctgg 5220

ccttgtaatt acaatgccac gcaacacttt gattataatt cttactgtgc aggtgacttg 5280

gtttgtcaag cctgttttga cggtcaagac tccctacatt tgtatccgca tttacgtgtt 5340

aatcagcagc cccttcagac cactgactac actgtttatg cgctttcact aatactacta 5400

ttagctaaca tgactcttgt catgggcacg ctaatagtta ctttctttgt gaacttctat 5460

ggtgtgcaaa taccatttta tggtacactt ttgatagatt atcaatccgc actggtgatt 5520

actttctcag tgtactactt ttataaggta atgaagtttt tccgccatct cacacatgga 5580

tgtaaaattc caacgtgtgt ggtatgtgcc aaacttcgta ccccacctac tataacagtt 5640

gagactgtcg ttcagggcag gaaataccca tctgttattg aaacaaatgg cgggtttaca 5700

atttgtaaag aacacaactt ctattgcaag gactgctctt tacaaacacc cggcactttc 5760

atcccgacag aagctattga gtcgctctca cgagctacca ggcttagtgt caaaccaaca 5820

gcaccagcat tcttacttgc tagagatgtt gagtgccaaa ctgatgttgt cgttgctcgc 5880

gcaatgcata accaaaatgc gcatgtgtgc atttcaaaat actctgatat ccgtaccgtt 5940

gaccaactac ttaagcctac tccactgttt tcatacactc ccgatgttat catcgcggca 6000

gactttgaca acagaggtag tcttaagaca gctaaagaat tagctgtggt tttgtcaatg 6060

gaccttaaac gtactataat tatcattgat caggcctatt ctagacctat tgataattat 6120

caggaagttg cttctcgtat tgagaagtat tacccagttg caaagatcac acccacaggt 6180

gacatcttta cagacattaa gcaagcgacc aatggccaag ctagtgactc tgctattaat 6240

gcagctgttc tggctgtcca gcgcggtctt gattttacaa ttgacaaccc taacaacata 6300

ttaccacatt acgcctttga cttttcgacc ctcaatgcag aagaccagtc taccattttg 6360

gagagtggtt gtgctaaagg caatctcaag ggcactaatg ttggtgttgt tctttcagct 6420

agccttgtta cacgtcttag tcagcaggct atacgtgtga ttgctaatgc tgcttcacgt 6480

aatggtgtta catgcgctgt tactccttct acacttgtta tgcgtgggaa tattgcaaca 6540

cagcccttga ctcgcatcaa agctggtgca cctcccatgc gtcaaaaaat tttatgtgtt 6600

atcctggcac ttgctattgt gtactttgct gctatggctt ttggcttttt ggcaagtcaa 6660

attacgctta atacagtgcc tacgattaaa tctgatatcc gcgcctctac cttctacgtt 6720

gttagagatg gagtcttgga tactgttcgt tcaaatgaca agtgctttgc aaataagttt 6780

ttggcatttg atagcttcat tcaagcacct tacactaatt cacctgactg tccagttgtt 6840

gtgggagttg ttgatgtaac gacgcactct attcctggaa ttccagcagg tgtcattcat 6900

agagacggtc tcatacttaa catttatgaa cagtctcttt atgaaactca tcagcgtcag 6960

tctatggtta gggatgcgtt gtcactcaag acagcaaatc tctttaacct aggcaagcgt 7020

gttgtagtag gatacactca acatgaagtt gttgtgggta cctcctattt taattctcct 7080

gcacttttta atgcaaagtg caccttctta cagtatcagg acactagaca actctattgc 7140

tatgatactg ttcctactga acataagctc tactctgatg tgcttccgca cgtcgagtat 7200

aaggctattg acattaatgg tgatcttgtt cctttcaaga taccagagca gataatgttc 7260

tatccacata ttgtgcgcta tactagcaat tcctattgcc gtatggggca ttgttttaat 7320

actaaccctg gtatttgcat ttcatttacg gacgaatttc cgtatagtga aaatgtcaaa 7380

cctggtgtgt actgtgctga tacctctttg cagttgtttt caaacctcgt tttgggcact 7440

gtatctggta ttcacatctt tacatcaaca gctgcattgc ttggatctac tattgtgatc 7500

atactatgcg ttgttgctgt tcttgcagtt cagcgattct tcaaggagta cacaactttt 7560

gttatgtaca cttgtggtct tgctcttgtc aacattgtag gcattgcact tatgtacaag 7620

tgccttgtct tcgcgatttt ctattatgca atctaccttt actttgtcct tactttcccc 7680

tcctttaaga ggaatgtggc attgttttac ttcgctgtag tgatcgtgcc gcacgtgagt 7740

aacatgcaat tgcttgcgct cattgtgtgt agcattatct actttctcta cacctatgtt 7800

catactgtag ctaagacagc tgggaaattt tcttccttct tagacgcagc taaagctact 7860

tttgtcattg acaatgaaaa gtacgtgttg cttaaagacc tcgctggtgc tgaatttgac 7920

cagtatctgg cctcttacaa caagtacaaa tatttttctg gtactgcttc tgataaggat 7980

tatgataagg tctgtatggc atttcttgcc aaggctttgt catcttttcg tgaaggaggc 8040

ggttcacagt tgtacacacc acctaaattt gcagttgttc agagtcttaa gaccaagctg 8100

caagcaggta tcaaaatcct cctgcaccct tcaggtgtag ttgagcgatg tatggtctca 8160

gttgtctaca atggatctgc attgaatggc atctggctta agaatgttgt ctactgccca 8220

cgccatgtaa ttggaaaatt ccgtggtgac cagtggactc acatggtctc aattgctgat 8280

tgccgcgact ttatagtcaa gtgtccaata cagggtattc agctaaatgt ccaatcagtt 8340

aagatggtag gagctctcct ccagttaact gttcatacca acaacacagc cactccagac 8400

tataagtttg aaaggctcca accaggatca tcgatgacaa ttgcttgtgc ttatgatggc 8460

attgtacggc atgtctatca cgtggtcctc caacttaata atcttattta tgcaagcttc 8520

cttaacggag cttgtggtag tgtgggttac actcttaagg gtaaaacact ctacttacat 8580

tacatgcacc acattgagtt taacaacaaa actcatagtg gtacagatct tgaaggtaac 8640

ttctatggcc cctatgtgga tgaggaagtt attcagcaac aaacagcatt ccagtattac 8700

actgataatg ttgttgctca attatatgca cacttactga ctgttgatgc tagaccaaaa 8760

tggctggcac aatctcagat aagtatcgag gatttcaact catgggctgc taacaattcc 8820

tttgctaact tcccatgtga acaaactaat atgtcctaca ttatgggact ctcgcaaaca 8880

gctcgagtcc ctgtagaacg tatcctcaat accattatac agctaaccac caatagagat 8940

ggtgcttgta ttatgggatc ttatgatttc gagtgcgatt ggacgccaga gatggtatac 9000

aatcaggctc caatttcatt gcagtcagga gtagttaaga aaacttgtac gtggttcttc 9060

cacttcttgt ttatggctat taccatgcta ctcgctgcca tgcatgtttt ccctgtacac 9120

ttgtatccaa tagtactgcc atgcttcact gtcgtggcat tcctgttgac tttaaccatt 9180

aaacacactg ttgtgtttac cactacatac ttgcttccgt cacttttgat gatggttgta 9240

aatgctaaca ctttttggat accgaacaca tttctgcgca cctgctacga aactatattc 9300

ggttccccaa ttgctcagcg actgtatggt tacactgttg ctctttatat gctgatctat 9360

gctggacttg caatcaacta tacgttgaaa acactccggt atagagcaac ttcattctta 9420

tctttttgca tgcagtggtt tcaatatggt tatgttgcac acattgcgta caaactgctt 9480

aataaaccct ggacagaatc actactcttc acagccttca caatgctaac cagtcatcct 9540

ttgttggctg ctcttagctg gtggctagct ggtcgcgtaa ctctgcccat tatcatgcct 9600

gacttagcta ttcgtgtttt ggcgtataac gtcattggct atgtcatatg tgttcgattt 9660

ggccttatgt ggcttgcaaa tcggttcaca actgtaccta tgggcacata ccagtatatg 9720

gtgtctgtag agcaacttaa gtacatgatg gcagttaaga tgtccccacc gcgtaatgcg 9780

tttgaggtgc ttatagccaa cattagactt cttggtttgg gtggaaaccg taacattgct 9840

gtttctactg tccaaaacaa aattcttgat gcaaaagcta ctgctgttgt tgttgctaac 9900

cttcttgaaa aggctggcgt cacaaacaag cacgctattt gcaaaaagat tgtgaaactc 9960

cacaatgata cccttaaagc caccacttat gaggaggttg aggtagcact tgtgaaactt 10020

ctttctcaca taattgagtt cttgccaact gatcaggtag atgcttatct agctgatgcg 10080

gccaatgctc aacatgttaa tacctatttt gacaacttgc ttgagaacaa agctgttgtt 10140

caggctgttg ccgatatcaa cattaatctg gattcttata gaatttataa ggaggcagat 10200

gctatttata aacgatctgt tgagatgaac gaatctccgc aggagcaaaa gaaaaagctt 10260

aaagctgtta acattgcaaa ggcggaatgg gagcgtgagg ctgcttctca gcgtaagctt 10320

gaaaagcttg ctgatgctgc tatgaagtct atgtatcttg cagaacgtgc tgaggatcgt 10380

cgcattaagc taacctctgg acttactgca atgctttacc atatgcttag acgtcttgac 10440

tcagataggg taaaagctct gtttgagtgc gctaaggcac aaatcttgcc aatacatgct 10500

gtagtcggaa tttctaatga caaccttaaa gttattttta acgataagga cagctactct 10560

cattatgtag agggcaacac acttatacat aagggagttc gctacactat tgtgaagaaa 10620

ctctccttag ataatgcacc tattgaaggc gtaccagaag aattccctgt ggtcgttgag 10680

actgttaggg aaggtgtgcc ccagttgcaa aataatgagc tatgtttgcg caatgttttc 10740

actgctcaga acacagctca ggacttcaat ggcaatgaat ccactgtaaa atctttttat 10800

gttactagaa ccggtaagaa gattttggtt gccattacat caactaaaga caatcttaag 10860

actgtgacct gccttactga gaccggtaag acagtcctta acttggaccc ccctatgcgc 10920

ttcgcacata ccgtaggtgg aaaacagtct gttgtctatc tctattttat tcagaatatt 10980

agttcactca acagaggtat ggttattggc cacatctctg aaactactat ccttcaggca 11040

agtggcactc aaattgagta ccagcaaaat gcctctcttt tgacctattt ggctttcgct 11100

gtagacccta agacagccta ccttaagcat cttgctgatg gtgggtctcc tatacagggt 11160

tgtattcaga tgattgctac tatgggtcct ggatttgcag ttactactaa accacaacct 11220

aatgagcatc agtattctta tggtggtgct tcaatttgtc tttattgccg tgctcatata 11280

ccacatcctg gtgttgatgg acggtgcccc tacaaaggcc gctttgttca catcgacaaa 11340

gataaggaac ctgtttcctt cgccttgact catgagccat gcagttcttg tcaacggtgg 11400

gttaattatg actgcacctg cggatctagt ctgcagaatt cggcttattt aaacgagtaa 11460

cgggttctag tgacgcccgg ctagaacccc tgcagcctgg aactcaacca gatgctgtaa 11520

aaagggcctt ccatgtgcat aatgatacca cctctggtat attcttaagc acaaaatcta 11580

actgcgctcg gtttaaaacc acacgcagtg ccctgccttt acctaataag ggagaggttg 11640

aattgtactt tgttactaag cagtgtgcag ctaaagtctt cgaaatcgag gaggaatgct 11700

acaacgctct tagtacagag ctttatacta ctgatgatac atttggtgtc cttgccaaaa 11760

ctgagttctt taagtttgac aagataccta atgtcaatcg ccagtatctg actaaatata 11820

cactcctgga cttggcttat gctctacgtc atttgtcaac atctaaggat gttattcaag 11880

aaatcttgat caccatgtgc ggaacccctg aagattggtt tggggaaaat tggtttgatc 11940

caattgagaa cccatccttt tacaaggagt tccataaact tggggatatt cttaaccgtt 12000

gtgttcttaa tgccaataag tttgctagtg cctgtataga cgctggtctt gttggcatat 12060

taacacccga caaccaagac ctcctgggtc agatctatga ctttggagat tttattatta 12120

cacaaccagg taatggatgt gtggacttag catcctatta ttcttattta atgcccatta 12180

tgtccatgac tcacatgtta aagtgtgagt gtatggatag tgatggcaac ccacttgagt 12240

atgatggatt tcagtatgac ttcacggact tcaagcttgg cttgttcgag aagtatttta 12300

agtactggga ccgtccttac catcctaaca ctgttgaatg tccagatgac cgttgcgtat 12360

tgcactgtgc gaacttcaat gtgttgtttg ctatgtgtat acctaatacg gcatttggca 12420

atctttgttc aagagctact gttgatggcc accttgtggt ccagacagtg ggtgtacact 12480

tgaaagaact cggtatagtc cttaaccagg acgttaccac acacatggca aatattaatc 12540

taaacactct attgcgattg gttggtgatc ccaccactat tgcaagtgtc tcagacaagt 12600

gtgtagattt aagaactcct tgtcagacct tggctactat gtctagcgga attgctaaac 12660

agtcagtcaa gcccgggcat tttaatcaac acttctacaa gcatttgctt gatagtaacc 12720

tattagacca acttggaata gacattcgcc acttctacta tatgcaggat ggtgaagcgg 12780

ctatcacaga ctacagctac tacaggtata atacccccac gatggtagat atcaagatgt 12840

tcttattttg ccttgaggtg gcagataagt atcttgagcc ctacgaaggt ggatgtatta 12900

atgcacagtc agttgtggtc tctaatttgg acaagtcagc gggctacccc tttaacaagc 12960

taggtaaggc tcgtaactat tacgacatga ctcatgccga gcaaaatcaa ctgtttgagt 13020

atacaaaacg caatgttttg cctacactca ctcagatgaa ccttaagtat gcaatttcag 13080

ccaaggatcg tgctcgcact gtggcaggag tgtctataat tagcaccatg actaacaggc 13140

agtaccatca aaagatgctg aaatctattt cacttgcacg caatcagacc atcgtgattg 13200

gaacaaccaa attctatggt ggttgggaca acatgttacg acgactgatg tgtaatatca 13260

acaatcccat tttagtgggt tgggattacc ctaagtgtga tcgttctatg ccaaacatgc 13320

tgcgcattgc cgcttcgtgc ttgctagcac gaaaacacac ttgctgtaac caaagccagc 13380

gattctaccg tttggctaat gaatgttgcc aagtactatc tgaagtggta gtctctggta 13440

acaacctcta tgtaaaacca ggtggcacta gcagtggtga tgcaaccaca gcttatgcca 13500

attcggtatt taacatctta caggtggttt ctgctaatgt agccaccttc ttatcaactt 13560

ccaccacgac acatcttaat aaggacattg cggacttgca tcatagtctt tatgaagata 13620

tttatcgtgg tgactctaat gatatcactg tcatcaatag attctaccag catctccaaa 13680

gttactttgg acttatgata ttgtctgatg atggtgtcgc atgcatagac tcagccgttg 13740

caaaggctgg agctgttgct gatcttgatg gtttccgaga cattttgttt taccaaaaca 13800

atgtttacat ggcagactca aagtgttgga cagaaactga catgaatgtt ggccctcatg 13860

aattttgctc acagcatact gtgttagcag agcatgatgg taaaccttac tacttacctt 13920

acccagatgt ctctcgcatt ctgggtgcat gcatctttgt ggatgacgtt aacaaggctg 13980

accctgttca gaaccttgaa cgttacatct cacttgcaat tgatgcatat cccctcacca 14040

aggttgaccc tattaagggt aaagtctttt atttgttact agactacata cgtgttcttg 14100

ctcaggagtt acaggatggt atccttgatg ctttccaatc actcactgac atgtcgtatg 14160

taaataactt tatgaatgag gccttttatg ctcagatgta tgagcaaagt cctacactac 14220

aggccagcgg tgtttgtgtg gtgtgtaatt cacccactat actgcgctgt ggtgattgca 14280

ttcgtcgacc actactttgt tgcgtctgtg cctaccagca tgttacgcag actacacata 14340

aacgtatcat tgctatcaac aactacatct gtagtgttga gaattgcaat gaggacaatg 14400

ttgaaaaact tttcatttct ggcactgcga tttattgtga gaatcacaaa cctacgctgt 14460

gcatacccat tgtagctaac ggttctgttt ttggtatcta tcgccacact gcccgtggta 14520

gtgatgacat agacctcttt aacgagcttg ctacatctaa ctatgacact attgaacctt 14580

atcagaaggc caatcgtgca cctttatcac ttatgctctt cgctgctgaa accattaagg 14640

cactcgagga gtctatcaag aagtcatatg ctaccgcaac cgtcaaggat gtgtatgacc 14700

aacgcttcat taaacttcta tgggaacagg gtaaaaagcc gccacccata acgaagaacc 14760

acattttcac tggctaccat tttaacaaga atggaaaaac ccaagttggt gattacattc 14820

ttgctaaaac agatggcagt gacacttata cttacagagg aacatctacc tacaaactcc 14880

aaacaggtga tgttctagtc ttaatggcac atgttgttac accgctctca gcaccccctg 14940

tgctaacgca gacaacatat gtcagaaaat cacttttacc cgactctgtt ggtgcgtctt 15000

attatgtgca acattttaag tcatataatg agatagctat gcagagggtt acaacagtat 15060

taggtccacc aggcacaggt aagtcaacct ttgctattgg tttggctaag tactttccca 15120

gtgcacgtat ttgctacact gcgtcttcgc atgcagcaat cgatgcactc tgtgaaaaag 15180

ctttcaagac aatacctgta ggccaatgca gtcgtatcgt acccacacgt acaactgttg 15240

agtgctttca ggagtttgtc gtaaataaca caactgcaca gtatatcttc tcgactatca 15300

atgccttacc tgacattaag tgtgacattg tagttgtaga tgaggtttct atgttgacca 15360

attatgagct ttcctctgtg aatgctcgtt tggtttacaa tcacattgtg tatgttggtg 15420

atccttatca gttaccttca cctagaacta tgcttacgtc tggccagctt tcgccagctg 15480

actataacgt agttactgat ataatggtac atgcaggagc ggatgttatg ctcgacatgt 15540

gctacagatg cccacgtgaa atcgttgaga cagtgtctaa acttgtctac gataacaaac 15600

taaaagcggc gaaaccgaac tcaagacagt gttacaagac cattgtgaac tttggtcctg 15660

gagacgttgc tcatgaggga caatctgcct acaacgaagc acagttgcgt ttcgcactcg 15720

catttagaca acaaaagcgg tgggataacg tgactttcat atctccatat aatgctatga 15780

atgtgaaagc atccttagca ggtttctcta ctcagaccgt tgactcttct caaggttctg 15840

agtatgatta tgttatcttt tgcgtgacca ctgattcagc acacgcactt aacatggctc 15900

gtttgaacgt tgcccttaca cgtgcaaaga taggtatcct tgtggtgttt aggcaggcaa 15960

acgaacttta caatagtttg cagtttgaat ctattgattc acagcttcag tcgagtgctg 16020

agaaaaacct cacaccactg tttaagcgct gcggctatga gtataatggc gtccatccag 16080

ctcatgcttt gacctggcat gattgtggtg cagagtaccg ctgtgaggag ccacttgcta 16140

aattagtagg agttgccgat ggcactctta tatcatacaa aaccctagta tccacacttg 16200

ggtttcttcc atcacttaaa attgatgcat atcataatat gttcctaaca cgtgatgcgt 16260

gtcgcaccta tgttcagagt tggatcggca tagatgttga agcagcacac gccataaaac 16320

ctaacaccgg gactaacctg ccattgcaaa taggttttag taccggaaag aatttttcag 16380

tcactccaga gggaatttgg gtaaacgagc acggatcttg cactgagccc gtccctgcca 16440

aaatacctcc tggagaacaa tttcgtcacc ttaaaaagga catgcgccag gcgcgtcctt 16500

ggatggttgt tcgacgtgag attgctactc acattgctga ggtagctcct cacactgatt 16560

atatatgctt tgtcacttgg gctcaccagc ttgagctagc gacaatgcgc tactttgtca 16620

aactaggtat ggaagagaga tgcttttgtg gcaggcgggc ttgtttcact aatggaactg 16680

agttcgcttg caaagcacac cattctctca ccattccaca atgtgattat gtgtacaatc 16740

cattcctcat cgacgtggct acgtggggat tctcgggacg gctttccacc aaccatgacg 16800

cggtgtgcac atatcatgct aatgcccatg ttgcatcagc tgatgcaatc atgacggtat 16860

gtttagctat ccatgaactg ttcagtactg ttgactggaa ctttgaattt ccagtaactg 16920

ctgagcaatc gcaacttaac aaggcctgtc gcttagtaca ggcgaattac ttaaatatac 16980

tactcactac aaccaaagcc acggtggttc acgatattgg taacccaaaa ggtatcccta 17040

tcgtgcgcaa acctggtgtt aaatatcact tctatgatca agcacccatt gtcaaacacg 17100

ttcaaaaact aaagtacaag ccagagatgg aggcccgttt caccgatggt ttgactatgt 17160

tttggaattg taatgttgac acataccctg ctaacgccct tgtgtgccgc tacgacactc 17220

atcggcagaa gcatttaatt ggacctaatg gttcagcact atatgttaat aagcatgctt 17280

ttctcacccc tgagatgcat acttatgcta cacataaact caacttggct ccactcatct 17340

actactccac cacagattgt agtagtgaac agcctattgt tgttacctac agagattgtg 17400

tcacccggtg caatactgga aaaactctct gtccaaatca tgctcttgaa tatcaagagt 17460

ttatcaatgc atacaatctc atggctcgcc atggatttaa tgtttacata ccacgcaatg 17520

tcaacgttta caactgttgg cttactttca ctaatctcca aaaccttgaa aacttagctt 17580

acaactgtta ttataagaac tgcaatgctc acgttgatgg gcagcttgat gtagttatta 17640

ataataacgc tgtatatgct aaggtcgaca ataatcttgt caaacttttc gacaaccgca 17700

ctaacttacc tgtctcagtg gcctttgaac attacactaa caggcatacc cgttcactgc 17760

caactacaca gctgttatct ggtttaggcg taaccgccac cagaaatttc actgtgtggt 17820

tcgacaatga tacaattttc caatacacta ttaatgtatc tacgtatact gacatcgacc 17880

ctagtaccca tgttgtcctc tgtgatgata ggtacggaac agattggagt cagtttaacc 17940

aacttcctaa tgcagtattc ctcaccaaaa ctaaggtgaa gaaaacagaa ccgtttgttt 18000

gtacagcact gaccctaaat ggcgtcgcca ttgacggtga agagctatac atctatgtac 18060

gctataacaa tcaactgacc acatttgcta ctacttgtac acagggtaga aatgttgagc 18120

agtttatacc taaaacacct atggaaagag acttccttga gatgtctcaa cagtccttca 18180

tccagcaaca tcaattgcag gaactgggtg ttgaacacat tatctatggt gatgattcca 18240

gtccagtcat tggcggaact cacacactta tctcactagt taaaaacaag tttgaacatc 18300

agcttgtcaa ccatgtttac aacccagtcc agaactgtgt tgttacctca cctaacgcaa 18360

gctccaagaa cgtttgcact gttcttgatg ttctacttga tgactacatt gacatcataa 18420

gacaagcaca tgccagttac acaagtaaat ccaaagtatt cactgtgtca attgacaacc 18480

aacaaattag attcatgctt tggcatgatg agcaagtcaa gacttgctac ccaatcttac 18540

agtcacttac caatggttac cagatgccat ctgtgtacaa aacattggtt actgacttac 18600

aaccagctga catccctaat tatcattcct acaccccccg ggtgcctgga gtagttaaga 18660

atgttatcaa gtaccgccaa cttttcaact acatagttaa aaaggatagg ttggcagtac 18720

cacacaatat gaccgtatta caccttggag ctgcatctgc actaggtaca gcaccaggtt 18780

cttcagtcat aaaacaaatg tttcctgaag gaactgttct tattgacctc gatataagag 18840

agttcacttc agatgctaac caaataatag ttacagacta cagaacttac ataccaccac 18900

accacgtaga cgtcatattt tctgacctct actgttgtga tgacatacac ttctttgaca 18960

atctaataag gatagttaag gagaggctcg ccctcggtgg ttctatcttt gttaagataa 19020

ctgaacattc attctcaccc gaactctact cacttgcggg ttggttcgat gattatcaac 19080

tattttgcac agcagtcaat gcctcgtctt cagaagcatt tttatgctgt tttaattatt 19140

tggggcttgc taaggaaaac attaatggtt ttaacttaca tgcttcctat attcaatggc 19200

gcaatgaaat agcgttgaca ccaacctatt ctcctttagc ggacaacccg gctacggcct 19260

gtaagctaaa agcaacgcct attatctcgg ctcgtgagtt agagaagaag cctattcttc 19320

gctatctcgt tgcatcaggg cgccttcttg tgaggccacc agaatgcaga gagctctatt 19380

gattatgacc ttactttgtc tcgttcgagc aaagtttgct gatgatctac tcgacttgct 19440

caccttcccg ggtgcacatc gcttcttaca taaacccacg aggaattcca gcagtctcta 19500

ctcgcgggct aataataatt ttgatgttgg cgttcttcct ggctacccca ctaagaacgt 19560

taacctcttc tcaccactta ctaactccac tttgcccatt aatggccttc atcggagtta 19620

ccaaccactc atgctgaatt gtcttactaa aataactaac cacactctca gcatgtatct 19680

cctacctagt gagatacaaa cttatagctg cggcggtgcc atggttaaat accagacaca 19740

tgatgcagtt cgtatcattt tagacctcac tgccactgac cacatctctg ttgaagtcgt 19800

tgaccaacat ggtgaaaatt atgtgtttgt ttgcagtgag cagtttaact acaccactgc 19860

attacacaat tctaccttct tctcacttaa ttctgcgctt tattgcttta ctaataacac 19920

ctacttaggt attcttccac ctgatttaac tgactttacg gtctaccgta ctggtcagtt 19980

ctatgctaat ggttaccttt taggtacttt acctattacg gttaactatg ttaggttgta 20040

tcggggtcat ttgtctgcca atagtgccca ctttgccctt gcaaacctaa ccgatacact 20100

cataacactt accaatacta ctatatcgca aatcacttat tgtgataagt cagtagttga 20160

ttcaatagca tgccagcgct cttctcacga agtggaggat gggttttact ccgaccctaa 20220

atctgccgtt agagctaggc aacgtactat tgttacacta cctaagctcc ctgagcttga 20280

agtagtgcag ttaaatattt ctgcacacat ggattttggc gaagccagac ttgacagcgt 20340

taccatcaat ggtaacacat cctattgtgt cactaagcct tacttcaggc ttgaaactaa 20400

ctttatgtgt acaggttgca ctatgaatct gcgcactgat acctgtagtt ttgacctgtc 20460

agcagtaaac aatggcatgt cattctctca attctgtcta agcactgaat ctggtgcttg 20520

tgagatgaaa attattgtta cctacgtatg gaattacttg ctaaggcagc gtttgtatgt 20580

tactgctgta gagggccaga ctcacactgg aaccacttca gtacatgcaa cagacacttc 20640

tagtgtaatc actgatgtct gcactgacta cactatctat ggagtctctg gtactggcat 20700

tattaagcca tcagatctct tattgcacaa tggcatagca ttcacctctc caacaggtga 20760

gctttatgca tttaaaaata taaccactgg caaaaccctt caggtcttac cgtgtgaaac 20820

cccttctcaa ctgattgtga taaacaacac cgttgtcggt gctatcacat ccagtaattc 20880

aactgaaaat aataggttta ctactactat tgtcacacct actttctttt attccacaaa 20940

tgccaccact ttcaactgca ctaagcctgt tttgtcctat ggacctatca gcgtgtgtag 21000

tgatggtgca attgtgggaa catccacatt acagaatact cgaccatcca tagttttact 21060

atacgatggc gaagttgaaa taccatctgc attttctctt tccgttcaga cggagtactt 21120

gcaagttcaa gcagagcaag ttatagttga ttgtcctcag tatgtatgca atggcaacag 21180

ccgttgtcta caattactgg cacaatacac ctcagcttgc tctaacattg aagcagctct 21240

gcattcctct gcacagttgg atagcagaga gattataaat atgtttcaaa catcaacaca 21300

gtccttgcag ttagctaata ttaccaactt caagggtgac tacaatttta gcagcatact 21360

aaccaccaga attggtggca gatctgctat tgaagacctt ctttttaata aagttgttac 21420

tagtggcctt ggcactgttg atcaggacta caaatcctgc tctagagaca tggccatcgc 21480

tgacttagtt tgttcccagt attacaatgg catcatggtt ctacctggtg ttgttgatgc 21540

tgagaaaatg gcaatgtata ctggctctct tactggagct atggtatttg gaggactgac 21600

tgccgcagcg gcaataccat ttgccacggc agtacaagct cgcctcaatt atgtcgcact 21660

gcaaacaaat gtactacaag aaaaccagaa aattcttgca gaatcattta accaagcagt 21720

tggcaatata tcacttgcac tatcttctgt taatgatgcc atccagcaaa cttctgaggc 21780

tcttaacacc gtagctattg ctattaaaaa gattcaaaca gttgttaacc agcagggcga 21840

ggcattatca cacctgactg cacagctgtc aaacaatttt caagcaattt cgacttctat 21900

tcaagacatt tacaaccgtc ttgaggaagt agaggctaac cagcaagttg accgtctcat 21960

cacaggacgg ttggctgcac ttaatgcata tgttactcag ttactcaatc agatgtctca 22020

gattagacaa tctcgattgt tagctcagca aaagattaat gagtgtgtca aatcacagtc 22080

gtccagatac ggtttctgtg gaaatggcac acacatcttc tcacttacac agactgcacc 22140

aaatggcata tttttcatgc atgcagtgct agtacccaac aaattcacac gtgtcaacgc 22200

ttctgccggc atttgtgtgg ataatacgag aggctactca ttgcagcctc aacttatact 22260

ctaccagttt aataactcct ggagagttac acctagaaat atgtatgaac ccagactgcc 22320

ccggcaggct gatttcatac aattaactga ttgcagcgtt actttttaca acaccaccgc 22380

tgctaatctt cccaatatta tccctgacat tatagatgtc aatcaaacag tcagtgatat 22440

tattgacaat ttacctacag caacacctcc tcagtgggat gttggtatct ataacaacac 22500

tattctcaac ctcaccgttg agattaatga tctacaagag cggtctaaaa acctctcaca 22560

gattgcagat cgtttacaaa attatattga caatcttaac aatactctag ttgaccttga 22620

atggctcaac agagtggaaa cttaccttaa atggccgtgg tatatatggc ttgccattgc 22680

cctggctctt attgcatttg tgacaatcct cataacaatc tttctttgta ctggttgttg 22740

tggtggttgc tttggttgtt gtggcggttg ttttggcctt ttctctaaga agaaaaggta 22800

taccgacgac caaccaacac cgtcctttaa gtttaaggaa tggtagtcga cgactgggcc 22860

gttaccatcc ctggacaata tattattgct atactagttg tcatctgcat tggtgtggca 22920

ctacttttta ttaatacttg cttagcttgt gttaaattat tttacaagtg ctacctaggg 22980

gcagcatatc ttgttaggcc tattatagtg tactactcca agccgaaccc cgtacctgag 23040

gatgagtttg taaaagtaca ccaatttcct agaaacactc actatgtctg acgcagaaga 23100

gtggcaaatt attgttttca ttgcgatcat atgggcgctt ggcgtcatcc tccagggagg 23160

ctatgccacg cgtaatcgtg tgatctatgt tattaaactt attctgcttt ggctgctcca 23220

acccttcacc ctagtggtga ccatttggac cgcagtcgac agatcatcta agaaggacgc 23280

agttttcatt gtgtccataa tttttgccgt actgaccttc atatcctggg ccaagtactg 23340

gtatgactca attcgtttat taatgaaaac cagatctgca tgggcactct cacctgagag 23400

tagactcctt gcagggatta tggatccaat gggtacatgg aggtgcattc ccatcgacca 23460

catggctcca attctcacac cagtcgttaa gcatggcaag ctcaagctac atgggcaaga 23520

gctggccaat ggcatatcag tcagaaatcc gccacaggat atggtgatag tgtcaccaag 23580

tgacaccttt cactacactt ttaagaaacc tgtggaatca aacaacgatc cagaatttgc 23640

tgttctgata taccagggtg accgcgcttc aaacgctgga cttcacacca taaccacttc 23700

aaaggccggt gacgctcgcc tgtataagta tatgtaatgt gcaactgcca tctgcagctg 23760

cgagatttat atagattgtg caataagcgg cacatcagaa gagaggatgt tcctgagctt 23820

attgaccctc tcgttaaaac tcgctgtttt gcttacagtc tcgtggttct tgctaatgct 23880

aatccaattg catttagcat actacctcgg aaaattctta tcaatggtga gcctttactg 23940

cttgaatatg gtagcatata tggtaaagac tttatcattc gaccatcgct ccaagtcatt 24000

cttgaagatg aattaaatta aagttttgac accaatctat catggctgca ccagtagtcc 24060

ctactactga cgcgtcttgg tttcaggtgc tcaaagctca aaacaaaaag gctactcatc 24120

ctcagtttcg tggcaatgga gttccgctta actccgccat caaacccgtt gaaaaccatg 24180

gctactggct gcgttacacc agacaaaagc caggtggtac tcccattcct ccatcctatg 24240

ccttttatta tactggcaca ggtcccagag gaaatcttaa gtatggtgaa ctccctccta 24300

atgatacccc agcaaccact cgtgttactt gggttaaggg ttcgggagct gacacttcta 24360

ttaaacctca tgttgccaaa cgcaacccca acaatcctaa acatcagctg ctacctctcc 24420

gattcccaac cggagatggc ccagctcaag gtttcagagt tgaccccttc aacgctagag 24480

gaagacctca ggagcgtgga agtggcccaa gatctcaatc tgttaactcc agaggcacag 24540

gcaatcagcc caggaaacgc gaccaatctg caccagctgc ggtacgtcgt aagacccagc 24600

atcaagctcc caagcggact ttacccaagg gtaaaaccat ttctcaggta tttggcaacc 24660

ggtctcgtac tggtgccaat gtcggctctg cagacactga gaagacgggt atggctgatc 24720

ctcgcatcat ggctctagcc agacatgtgc ctggtgttca ggaaatgctt tttgctggcc 24780

accttgagag caactttcag gcgggggcaa ttacccttac cttctcctac tcaatcacag 24840

tcaaggaggg ttctcctgac tatgagagac ttaaggatgc gctcaatacg gtcgttaacc 24900

agacctatga gccacctacc aaaccaacta aggacaagaa gcctgacaaa caagaccagt 24960

ctgctaaacc caaacagcag aagaaaccta aaaaggtaac tctgccagca gacaaacagg 25020

attgggagtg ggatgatgct tttgagataa agcaggaatc agcagcgtag acatcaatct 25080

atgtctgtta aacccaccca actccactca aatatctctt tggttccaga gagtcgtagt 25140

gtatagccag agagccagtc agagggcgct atcatgcaaa ctagggctgg ctactctagc 25200

acagaatcac atcccgataa tcaacagtgc tagaaggttg attataccat ttaatatgcc 25260

gaggccacgc ggagtacgat cgagggtaca gcataatctc aacttttgtt gagccacaat 25320

tttaatccta attggagaag gccaaaggac tgtactactt ttgtgggtgt agcagtcgcc 25380

cagtgggaaa gcgccaacta ggttacaatt gtggtgggga caaatt 25426

<210> 6

<211> 25426

<212> RNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus BIV-Isolate 2.0307 ssRNA + Genome

<400> 6

gauugguguc gguagagaac uagcgaagcu agggagauaa aauuauagac uaaugcuaua 60

auuuuuaucu uuagucuaua auuuuaucuc ccuagcuucg cuaguucucu accgacacca 120

auccaggugc gucugccacc aaguuggcua cccuuccuag gggcgcuuuc gcgcuugcuc 180

accauuagau uaccuggaaa ccagccauuc agguuggagu uuccccaggc ucuuuugugu 240

gggcauuagc ggcuuguggu uuuugcacaa aaucuaagcu acuuaccguu ccucugacca 300

uccaccacuu cuauagacag cacugauuac cguaggguuu aagucacacc ggucugcacc 360

gcccgucagc ggacacauua cccagcauag cacuccuugc accgagccua gguaggauaa 420

aacccccuac cgggugacuc uuaaggcguu uccuccacgg gauagccacu agucacuagg 480

uguaagugau cugaucuggg cguauugugu ugcgcaagug ugauacccau aggagcgugg 540

aauccuauuc ugcggcucag ugccugauau agcugugaaa uggccaagaa caaguccaag 600

cgcgacgcua uugcguugcc ugaaaaugua ccaccaccuc ugcaacuuuu cauucauguu 660

gcagcugcug aagaggguca cccuaagguu acuacuuacc uuggcaacua uaaccucuau 720

gccaccaagg cuccgccugg cgugcagguu cuuagugcua aaaccucucu uacugacuuu 780

gagaaugucu uuggagcuca acccaccuug cgaucaauuc guaaucuggu uugugaggcu 840

cgcucggcug aauggacaac uuccaagaau gcuuuugcac ucaaagccac ucaacuugac 900

uacucugaug ccguuuugag ggcaaugauu cguuucugcc cuccaaaggu guccacacuc 960

gcugccuuug cucuuuuugg ccgauugguu aaaauugagg acaaggaacu ugcugaguua 1020

gcucgugaua cugcccuuga guuggcguac acggcuaaaa uugguacauc ucuugcugac 1080

acgagaucug ucucacuuau ucauaaggac gcuuaucuaa cucucaguaa ugagguuguu 1140

ggcguaacuu uuacugccgc acuuauggca aaggcuacca cuguuaaugg agcaaugcaa 1200

uacucaaacu uuuaccucua cccucgugcc acuauuaagg uaaccgaugg uaaggcugaa 1260

gcaauugcaa cuaagccucu gucugcugcc acuaaaggca agcaaaucac agaggauguc 1320

aaccuccucc cugacuauca gcagcugcuu guugaucaag ugacuggcac ugagguuaag 1380

guuggagcuc uaaccuaugu uaagaccacu gauucgccac cccuuuacuu ucccaaaguc 1440

aaggguggug uuauugguau ugcacuuaag cagcagggca cugcggcuaa gaagcucaau 1500

guagucuucc augcucaacc ugaugauguu cugcuagccu ucauacaacu ucagcaauuc 1560

uugaaccgua cuucggauuc aaguguugaa auuacugauu gccagaguua ugaaguaucu 1620

ccaacuguga cggucaaaau uggcccgucu aaaccugggg augucaucgu ggcuacugau 1680

gaggaauacc uuaaaugcuu ugaaaccccu gagguaggua ggcucuauaa gguuuuccaa 1740

acucaaucuu gggcuaucau ugagcgugcc uucuccaguu ugaagauccg cguguccaaa 1800

gcuuuaucag cauuuauaag uuuucugcaa aaccuugcag auaacuuuac ugcaauaagu 1860

gguguuguca cugcacucau ucgugaacuc caggaucuua cccuggaugu ggcgacacgu 1920

aucacuaaca uacaauuugu uuaccgugcc gguaagcuua uugucgacac gacaaguguc 1980

auagcuaaac uuuuccagcc auuuugugau uuuauaucac cuuuccuucg gaaaguugcu 2040

gguuuugcaa uuuacacugu ugguaaucgc augcuuaugu uuaccagcac uggcaccuuu 2100

cuucucacaa aggcaacuac uaagauacuc aauaaggcaa aguacaucuu ugauguggag 2160

ccugaguacc caguagaugu aacaacaucc aaaguuguag uacaugaagc acuccagcaa 2220

accgacacua agccuacuag agcucuggag gcuguugaug ucguuguugg uaauacugua 2280

cugcaaaugg cuacugaugg cacugcguuc uacccaucgg augguacgca cgccucucuu 2340

ccaggauuca aagcagguuc ggaugagcuu uucauaagcu ucaacugcga ccucuuugau 2400

gaugagacua augcucaaau caacgaaaca cucgcugcau augagcuuaa ccaacuagug 2460

gcuccaggug auucuacacc gcgucaaauu gcgacguugg uugucgauac acuugcagau 2520

gcuauaacag accacuuucc ggagaaaacc auugaucuac cugaagacua ucaagucuuu 2580

ucugaccaug augaccuccc acucgcacaa uaccacaucc cugaucaccu gagccuguau 2640

auucaggcua uggaagguga agaugauagu ggugaugaaa uauguauuga ggacgaugau 2700

uacgacuguc cucaagccga cgaagacaca gaaggaguaa uuccccaaca gugggaacuu 2760

ccugauguug auaaauuuuu acucaagauc caggaacgga agaccagcag cgacgaagua 2820

cuuagcgucg acgucuaucc uaaaccagag ccggucggca auguugggau ugacgacagc 2880

gcgucggaaa agaagccaaa uggggacuca guaccggauc cugaggucca uccaacacua 2940

gagagugugg auguugaacg accaaccgaa acagcaaacc aggcuguuga agacaaaccu 3000

ucugauacca ccuuuguggu ugaugaggaa caauuacaag aaucaacacc agaacaugaa 3060

cuccgcuccu augaagggga guuugauucu gaugaugaaa uuauuauucc uauaguacca 3120

guaacaccug cggauuuaaa accacagacu auuacuauaa aggaguacuu uaagucugaa 3180

aaacuugaga cuauuaacga aggauccaca gagucaguua cacaaucuga cgauucguuu 3240

gacgagucau uuguugaugc ugagucugau gauccacaag auccugcugu auaugaugau 3300

acaacaauua uaacggacag cacugaugua ggcgaugagc cugagacaac ucuagcuacc 3360

aucguuaaca caccucugac acucgauaau aacuugccac cugaagccau uaaacaaccc 3420

agcccaacua agguugaguu aguuguuggu gaauuggcga guauuaaauu ugacaauucu 3480

guucuaguca acccugcuaa ugcgcaauua acaaauggcg guggagcugc ucgugcaauu 3540

gcaaaauuag cugguccaaa auaucaagag uacuguaaua guguggcucc uaucucagga 3600

ccgcuuacca cggacucuuu ugaugccaag aaauuuggug uagccugcau cuugcaugua 3660

gugccaccca aagguucuga cccuaaugua caagaacucc uguaucaagc uuacaagagu 3720

auccuuacug aaccagcaca cuauguuaua ccuauacuag gugcugguau cuuuggaugc 3780

aauccagucc acucucugga ugcguucagg aaagcauguc caagugacau aggucguguc 3840

acccuuguca cuaugaacaa aaaccauuug cagguguggg augcucucaa uaggaccauu 3900

guacgcacca cuacugacua ugaucaaguu accaccaagg cccuuacacc ccagggagug 3960

uuagaagcca aucucuuuga uggugaggac uuuguucaag aaccaaaacc cggucaaauc 4020

uaccuugagg uuacugaaga aguucagaac caagccaagg aacuugaccu uaaccuucag 4080

caauacugcg ucuaccugaa gacuugccac cauaaauggg uugugagucg uacgaacggg 4140

uugaugcauc uaaaacaaaa agauaacaau uguuuuguua gugcaggugu aaaccuguuu 4200

caaaacacug cuuaucaacu uagaccugcu auugaugcuc ucuauaggga guaucuuaau 4260

gguaauccaa auagauuugu ugcuuggauc uacgcaucca cuaaccgucg uguuggugag 4320

auggguuguc cacagcaagu uauuucuuug cucguuagua acucugacgc agcauuuuca 4380

gcaacuacag ccuguuguaa caccuacuuu aaccacacag guguuauuuc aguagcucgu 4440

gaauaugacc caauacaacc aaaggucuac ugcaugaagu gugaugugug gacucccuuu 4500

acaccccaga guggaaaagg ugcaguugca auugguauuu cugcagauga accuaccggu 4560

ccugccauua aauuugccgc agcucacugc ugguacacua auggcaagaa aacaguuaau 4620

ggcuaugaca cuaaagcuaa uguuguagcu accuaucaua gguuugacgu gccuaagccu 4680

caacuugucg aggacguggu ugcgcugccu acuaaaaaug acuuugaagu ucucaauguu 4740

gaagaacugc cgcaggauag ugugcuccau uuggacccac cuccuguaca ggccuuacaa 4800

ccuaaggcua accaacacau ugagauucua gaaaacccag auuaucugga cauuuuggau 4860

cuuuggauuc guaaacccaa auucauccuc guaaagucgu ggaguguuuu ggguagagca 4920

cuauguaagg cagguaaagu ugucuuuguc agugcuucgc uuuugacccg uuucuacaau 4980

uaccuuguag agauuggugc ucuugacuca acaauaaggu ugucagucga ucuuaccugu 5040

aaauuuguua gaacgguucu cccaucgucu aacacuguac acaaaacuug ucuuggucug 5100

uauuauucag cccagacacu uuuuguuucu uuagcaccau uccuuauguu accagcugua 5160

guuagucugc uuaauucagg cuauacaauu ggcacauauu uguaugcaaa aacuggcugg 5220

ccuuguaauu acaaugccac gcaacacuuu gauuauaauu cuuacugugc aggugacuug 5280

guuugucaag ccuguuuuga cggucaagac ucccuacauu uguauccgca uuuacguguu 5340

aaucagcagc cccuucagac cacugacuac acuguuuaug cgcuuucacu aauacuacua 5400

uuagcuaaca ugacucuugu caugggcacg cuaauaguua cuuucuuugu gaacuucuau 5460

ggugugcaaa uaccauuuua ugguacacuu uugauagauu aucaauccgc acuggugauu 5520

acuuucucag uguacuacuu uuauaaggua augaaguuuu uccgccaucu cacacaugga 5580

uguaaaauuc caacgugugu gguaugugcc aaacuucgua ccccaccuac uauaacaguu 5640

gagacugucg uucagggcag gaaauaccca ucuguuauug aaacaaaugg cggguuuaca 5700

auuuguaaag aacacaacuu cuauugcaag gacugcucuu uacaaacacc cggcacuuuc 5760

aucccgacag aagcuauuga gucgcucuca cgagcuacca ggcuuagugu caaaccaaca 5820

gcaccagcau ucuuacuugc uagagauguu gagugccaaa cugauguugu cguugcucgc 5880

gcaaugcaua accaaaaugc gcaugugugc auuucaaaau acucugauau ccguaccguu 5940

gaccaacuac uuaagccuac uccacuguuu ucauacacuc ccgauguuau caucgcggca 6000

gacuuugaca acagagguag ucuuaagaca gcuaaagaau uagcuguggu uuugucaaug 6060

gaccuuaaac guacuauaau uaucauugau caggccuauu cuagaccuau ugauaauuau 6120

caggaaguug cuucucguau ugagaaguau uacccaguug caaagaucac acccacaggu 6180

gacaucuuua cagacauuaa gcaagcgacc aauggccaag cuagugacuc ugcuauuaau 6240

gcagcuguuc uggcugucca gcgcggucuu gauuuuacaa uugacaaccc uaacaacaua 6300

uuaccacauu acgccuuuga cuuuucgacc cucaaugcag aagaccaguc uaccauuuug 6360

gagagugguu gugcuaaagg caaucucaag ggcacuaaug uugguguugu ucuuucagcu 6420

agccuuguua cacgucuuag ucagcaggcu auacguguga uugcuaaugc ugcuucacgu 6480

aaugguguua caugcgcugu uacuccuucu acacuuguua ugcgugggaa uauugcaaca 6540

cagcccuuga cucgcaucaa agcuggugca ccucccaugc gucaaaaaau uuuauguguu 6600

auccuggcac uugcuauugu guacuuugcu gcuauggcuu uuggcuuuuu ggcaagucaa 6660

auuacgcuua auacagugcc uacgauuaaa ucugauaucc gcgccucuac cuucuacguu 6720

guuagagaug gagucuugga uacuguucgu ucaaaugaca agugcuuugc aaauaaguuu 6780

uuggcauuug auagcuucau ucaagcaccu uacacuaauu caccugacug uccaguuguu 6840

gugggaguug uugauguaac gacgcacucu auuccuggaa uuccagcagg ugucauucau 6900

agagacgguc ucauacuuaa cauuuaugaa cagucucuuu augaaacuca ucagcgucag 6960

ucuaugguua gggaugcguu gucacucaag acagcaaauc ucuuuaaccu aggcaagcgu 7020

guuguaguag gauacacuca acaugaaguu guugugggua ccuccuauuu uaauucuccu 7080

gcacuuuuua augcaaagug caccuucuua caguaucagg acacuagaca acucuauugc 7140

uaugauacug uuccuacuga acauaagcuc uacucugaug ugcuuccgca cgucgaguau 7200

aaggcuauug acauuaaugg ugaucuuguu ccuuucaaga uaccagagca gauaauguuc 7260

uauccacaua uugugcgcua uacuagcaau uccuauugcc guauggggca uuguuuuaau 7320

acuaacccug guauuugcau uucauuuacg gacgaauuuc cguauaguga aaaugucaaa 7380

ccuggugugu acugugcuga uaccucuuug caguuguuuu caaaccucgu uuugggcacu 7440

guaucuggua uucacaucuu uacaucaaca gcugcauugc uuggaucuac uauugugauc 7500

auacuaugcg uuguugcugu ucuugcaguu cagcgauucu ucaaggagua cacaacuuuu 7560

guuauguaca cuuguggucu ugcucuuguc aacauuguag gcauugcacu uauguacaag 7620

ugccuugucu ucgcgauuuu cuauuaugca aucuaccuuu acuuuguccu uacuuucccc 7680

uccuuuaaga ggaauguggc auuguuuuac uucgcuguag ugaucgugcc gcacgugagu 7740

aacaugcaau ugcuugcgcu cauugugugu agcauuaucu acuuucucua caccuauguu 7800

cauacuguag cuaagacagc ugggaaauuu ucuuccuucu uagacgcagc uaaagcuacu 7860

uuugucauug acaaugaaaa guacguguug cuuaaagacc ucgcuggugc ugaauuugac 7920

caguaucugg ccucuuacaa caaguacaaa uauuuuucug guacugcuuc ugauaaggau 7980

uaugauaagg ucuguauggc auuucuugcc aaggcuuugu caucuuuucg ugaaggaggc 8040

gguucacagu uguacacacc accuaaauuu gcaguuguuc agagucuuaa gaccaagcug 8100

caagcaggua ucaaaauccu ccugcacccu ucagguguag uugagcgaug uauggucuca 8160

guugucuaca auggaucugc auugaauggc aucuggcuua agaauguugu cuacugccca 8220

cgccauguaa uuggaaaauu ccguggugac caguggacuc acauggucuc aauugcugau 8280

ugccgcgacu uuauagucaa guguccaaua caggguauuc agcuaaaugu ccaaucaguu 8340

aagaugguag gagcucuccu ccaguuaacu guucauacca acaacacagc cacuccagac 8400

uauaaguuug aaaggcucca accaggauca ucgaugacaa uugcuugugc uuaugauggc 8460

auuguacggc augucuauca cgugguccuc caacuuaaua aucuuauuua ugcaagcuuc 8520

cuuaacggag cuugugguag uguggguuac acucuuaagg guaaaacacu cuacuuacau 8580

uacaugcacc acauugaguu uaacaacaaa acucauagug guacagaucu ugaagguaac 8640

uucuauggcc ccuaugugga ugaggaaguu auucagcaac aaacagcauu ccaguauuac 8700

acugauaaug uuguugcuca auuauaugca cacuuacuga cuguugaugc uagaccaaaa 8760

uggcuggcac aaucucagau aaguaucgag gauuucaacu caugggcugc uaacaauucc 8820

uuugcuaacu ucccauguga acaaacuaau auguccuaca uuaugggacu cucgcaaaca 8880

gcucgagucc cuguagaacg uauccucaau accauuauac agcuaaccac caauagagau 8940

ggugcuugua uuaugggauc uuaugauuuc gagugcgauu ggacgccaga gaugguauac 9000

aaucaggcuc caauuucauu gcagucagga guaguuaaga aaacuuguac gugguucuuc 9060

cacuucuugu uuauggcuau uaccaugcua cucgcugcca ugcauguuuu cccuguacac 9120

uuguauccaa uaguacugcc augcuucacu gucguggcau uccuguugac uuuaaccauu 9180

aaacacacug uuguguuuac cacuacauac uugcuuccgu cacuuuugau gaugguugua 9240

aaugcuaaca cuuuuuggau accgaacaca uuucugcgca ccugcuacga aacuauauuc 9300

gguuccccaa uugcucagcg acuguauggu uacacuguug cucuuuauau gcugaucuau 9360

gcuggacuug caaucaacua uacguugaaa acacuccggu auagagcaac uucauucuua 9420

ucuuuuugca ugcagugguu ucaauauggu uauguugcac acauugcgua caaacugcuu 9480

aauaaacccu ggacagaauc acuacucuuc acagccuuca caaugcuaac cagucauccu 9540

uuguuggcug cucuuagcug guggcuagcu ggucgcguaa cucugcccau uaucaugccu 9600

gacuuagcua uucguguuuu ggcguauaac gucauuggcu augucauaug uguucgauuu 9660

ggccuuaugu ggcuugcaaa ucgguucaca acuguaccua ugggcacaua ccaguauaug 9720

gugucuguag agcaacuuaa guacaugaug gcaguuaaga uguccccacc gcguaaugcg 9780

uuugaggugc uuauagccaa cauuagacuu cuugguuugg guggaaaccg uaacauugcu 9840

guuucuacug uccaaaacaa aauucuugau gcaaaagcua cugcuguugu uguugcuaac 9900

cuucuugaaa aggcuggcgu cacaaacaag cacgcuauuu gcaaaaagau ugugaaacuc 9960

cacaaugaua cccuuaaagc caccacuuau gaggagguug agguagcacu ugugaaacuu 10020

cuuucucaca uaauugaguu cuugccaacu gaucagguag augcuuaucu agcugaugcg 10080

gccaaugcuc aacauguuaa uaccuauuuu gacaacuugc uugagaacaa agcuguuguu 10140

caggcuguug ccgauaucaa cauuaaucug gauucuuaua gaauuuauaa ggaggcagau 10200

gcuauuuaua aacgaucugu ugagaugaac gaaucuccgc aggagcaaaa gaaaaagcuu 10260

aaagcuguua acauugcaaa ggcggaaugg gagcgugagg cugcuucuca gcguaagcuu 10320

gaaaagcuug cugaugcugc uaugaagucu auguaucuug cagaacgugc ugaggaucgu 10380

cgcauuaagc uaaccucugg acuuacugca augcuuuacc auaugcuuag acgucuugac 10440

ucagauaggg uaaaagcucu guuugagugc gcuaaggcac aaaucuugcc aauacaugcu 10500

guagucggaa uuucuaauga caaccuuaaa guuauuuuua acgauaagga cagcuacucu 10560

cauuauguag agggcaacac acuuauacau aagggaguuc gcuacacuau ugugaagaaa 10620

cucuccuuag auaaugcacc uauugaaggc guaccagaag aauucccugu ggucguugag 10680

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uuaaaccuca uguugccaaa cgcaacccca acaauccuaa acaucagcug cuaccucucc 24420

gauucccaac cggagauggc ccagcucaag guuucagagu ugaccccuuc aacgcuagag 24480

gaagaccuca ggagcgugga aguggcccaa gaucucaauc uguuaacucc agaggcacag 24540

gcaaucagcc caggaaacgc gaccaaucug caccagcugc gguacgucgu aagacccagc 24600

aucaagcucc caagcggacu uuacccaagg guaaaaccau uucucaggua uuuggcaacc 24660

ggucucguac uggugccaau gucggcucug cagacacuga gaagacgggu auggcugauc 24720

cucgcaucau ggcucuagcc agacaugugc cugguguuca ggaaaugcuu uuugcuggcc 24780

accuugagag caacuuucag gcgggggcaa uuacccuuac cuucuccuac ucaaucacag 24840

ucaaggaggg uucuccugac uaugagagac uuaaggaugc gcucaauacg gucguuaacc 24900

agaccuauga gccaccuacc aaaccaacua aggacaagaa gccugacaaa caagaccagu 24960

cugcuaaacc caaacagcag aagaaaccua aaaagguaac ucugccagca gacaaacagg 25020

auugggagug ggaugaugcu uuugagauaa agcaggaauc agcagcguag acaucaaucu 25080

augucuguua aacccaccca acuccacuca aauaucucuu ugguuccaga gagucguagu 25140

guauagccag agagccaguc agagggcgcu aucaugcaaa cuagggcugg cuacucuagc 25200

acagaaucac aucccgauaa ucaacagugc uagaagguug auuauaccau uuaauaugcc 25260

gaggccacgc ggaguacgau cgaggguaca gcauaaucuc aacuuuuguu gagccacaau 25320

uuuaauccua auuggagaag gccaaaggac uguacuacuu uugugggugu agcagucgcc 25380

cagugggaaa gcgccaacua gguuacaauu guggugggga caaauu 25426

<210> 7

<211> 3480

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus BIV-Isolate 2.0307 Spike Antigen cDNA

<400> 7

atgcagagag ctctattgat tatgacctta ctttgtctcg ttcgagcaaa gtttgctgat 60

gatctactcg acttgctcac cttcccgggt gcacatcgct tcttacataa acccacgagg 120

aattccagca gtctctactc gcgggctaat aataattttg atgttggcgt tcttcctggc 180

taccccacta agaacgttaa cctcttctca ccacttacta actccacttt gcccattaat 240

ggccttcatc ggagttacca accactcatg ctgaattgtc ttactaaaat aactaaccac 300

actctcagca tgtatctcct acctagtgag atacaaactt atagctgcgg cggtgccatg 360

gttaaatacc agacacatga tgcagttcgt atcattttag acctcactgc cactgaccac 420

atctctgttg aagtcgttga ccaacatggt gaaaattatg tgtttgtttg cagtgagcag 480

tttaactaca ccactgcatt acacaattct accttcttct cacttaattc tgcgctttat 540

tgctttacta ataacaccta cttaggtatt cttccacctg atttaactga ctttacggtc 600

taccgtactg gtcagttcta tgctaatggt taccttttag gtactttacc tattacggtt 660

aactatgtta ggttgtatcg gggtcatttg tctgccaata gtgcccactt tgcccttgca 720

aacctaaccg atacactcat aacacttacc aatactacta tatcgcaaat cacttattgt 780

gataagtcag tagttgattc aatagcatgc cagcgctctt ctcacgaagt ggaggatggg 840

ttttactccg accctaaatc tgccgttaga gctaggcaac gtactattgt tacactacct 900

aagctccctg agcttgaagt agtgcagtta aatatttctg cacacatgga ttttggcgaa 960

gccagacttg acagcgttac catcaatggt aacacatcct attgtgtcac taagccttac 1020

ttcaggcttg aaactaactt tatgtgtaca ggttgcacta tgaatctgcg cactgatacc 1080

tgtagttttg acctgtcagc agtaaacaat ggcatgtcat tctctcaatt ctgtctaagc 1140

actgaatctg gtgcttgtga gatgaaaatt attgttacct acgtatggaa ttacttgcta 1200

aggcagcgtt tgtatgttac tgctgtagag ggccagactc acactggaac cacttcagta 1260

catgcaacag acacttctag tgtaatcact gatgtctgca ctgactacac tatctatgga 1320

gtctctggta ctggcattat taagccatca gatctcttat tgcacaatgg catagcattc 1380

acctctccaa caggtgagct ttatgcattt aaaaatataa ccactggcaa aacccttcag 1440

gtcttaccgt gtgaaacccc ttctcaactg attgtgataa acaacaccgt tgtcggtgct 1500

atcacatcca gtaattcaac tgaaaataat aggtttacta ctactattgt cacacctact 1560

ttcttttatt ccacaaatgc caccactttc aactgcacta agcctgtttt gtcctatgga 1620

cctatcagcg tgtgtagtga tggtgcaatt gtgggaacat ccacattaca gaatactcga 1680

ccatccatag ttttactata cgatggcgaa gttgaaatac catctgcatt ttctctttcc 1740

gttcagacgg agtacttgca agttcaagca gagcaagtta tagttgattg tcctcagtat 1800

gtatgcaatg gcaacagccg ttgtctacaa ttactggcac aatacacctc agcttgctct 1860

aacattgaag cagctctgca ttcctctgca cagttggata gcagagagat tataaatatg 1920

tttcaaacat caacacagtc cttgcagtta gctaatatta ccaacttcaa gggtgactac 1980

aattttagca gcatactaac caccagaatt ggtggcagat ctgctattga agaccttctt 2040

tttaataaag ttgttactag tggccttggc actgttgatc aggactacaa atcctgctct 2100

agagacatgg ccatcgctga cttagtttgt tcccagtatt acaatggcat catggttcta 2160

cctggtgttg ttgatgctga gaaaatggca atgtatactg gctctcttac tggagctatg 2220

gtatttggag gactgactgc cgcagcggca ataccatttg ccacggcagt acaagctcgc 2280

ctcaattatg tcgcactgca aacaaatgta ctacaagaaa accagaaaat tcttgcagaa 2340

tcatttaacc aagcagttgg caatatatca cttgcactat cttctgttaa tgatgccatc 2400

cagcaaactt ctgaggctct taacaccgta gctattgcta ttaaaaagat tcaaacagtt 2460

gttaaccagc agggcgaggc attatcacac ctgactgcac agctgtcaaa caattttcaa 2520

gcaatttcga cttctattca agacatttac aaccgtcttg aggaagtaga ggctaaccag 2580

caagttgacc gtctcatcac aggacggttg gctgcactta atgcatatgt tactcagtta 2640

ctcaatcaga tgtctcagat tagacaatct cgattgttag ctcagcaaaa gattaatgag 2700

tgtgtcaaat cacagtcgtc cagatacggt ttctgtggaa atggcacaca catcttctca 2760

cttacacaga ctgcaccaaa tggcatattt ttcatgcatg cagtgctagt acccaacaaa 2820

ttcacacgtg tcaacgcttc tgccggcatt tgtgtggata atacgagagg ctactcattg 2880

cagcctcaac ttatactcta ccagtttaat aactcctgga gagttacacc tagaaatatg 2940

tatgaaccca gactgccccg gcaggctgat ttcatacaat taactgattg cagcgttact 3000

ttttacaaca ccaccgctgc taatcttccc aatattatcc ctgacattat agatgtcaat 3060

caaacagtca gtgatattat tgacaattta cctacagcaa cacctcctca gtgggatgtt 3120

ggtatctata acaacactat tctcaacctc accgttgaga ttaatgatct acaagagcgg 3180

tctaaaaacc tctcacagat tgcagatcgt ttacaaaatt atattgacaa tcttaacaat 3240

actctagttg accttgaatg gctcaacaga gtggaaactt accttaaatg gccgtggtat 3300

atatggcttg ccattgccct ggctcttatt gcatttgtga caatcctcat aacaatcttt 3360

ctttgtactg gttgttgtgg tggttgcttt ggttgttgtg gcggttgttt tggccttttc 3420

tctaagaaga aaaggtatac cgacgaccaa ccaacaccgt cctttaagtt taaggaatgg 3480

<210> 8

<211> 1158

<212> PRT

<213> Porcine Deltacorona Virus

<400> 8

Met Gln Arg Ala Leu Leu Ile Met Thr Leu Leu Cys Leu Val Arg Ala

1 5 10 15

Lys Phe Ala Asp Asp Leu Leu Asp Leu Leu Thr Phe Pro Gly Ala His

20 25 30

Arg Phe Leu His Lys Pro Thr Arg Asn Ser Ser Ser Leu Tyr Ser Arg

35 40 45

Ala Asn Asn Asn Phe Asp Val Gly Val Leu Pro Gly Tyr Pro Thr Lys

50 55 60

Asn Val Asn Leu Phe Ser Pro Leu Thr Asn Ser Thr Leu Pro Ile Asn

65 70 75 80

Gly Leu His Arg Ser Tyr Gln Pro Leu Met Leu Asn Cys Leu Thr Lys

85 90 95

Ile Thr Asn His Thr Leu Ser Met Tyr Leu Leu Pro Ser Glu Ile Gln

100 105 110

Thr Tyr Ser Cys Gly Gly Ala Met Val Lys Tyr Gln Thr His Asp Ala

115 120 125

Val Arg Ile Ile Leu Asp Leu Thr Ala Thr Asp His Ile Ser Val Glu

130 135 140

Val Val Asp Gln His Gly Glu Asn Tyr Val Phe Val Cys Ser Glu Gln

145 150 155 160

Phe Asn Tyr Thr Thr Ala Leu His Asn Ser Thr Phe Phe Ser Leu Asn

165 170 175

Ser Ala Leu Tyr Cys Phe Thr Asn Asn Thr Tyr Leu Gly Ile Leu Pro

180 185 190

Pro Asp Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr Ala

195 200 205

Asn Gly Tyr Leu Leu Gly Thr Leu Pro Ile Thr Val Asn Tyr Val Arg

210 215 220

Leu Tyr Arg Gly His Leu Ser Ala Asn Ser Ala His Phe Ala Leu Ala

225 230 235 240

Asn Leu Thr Asp Thr Leu Ile Thr Leu Thr Asn Thr Thr Ile Ser Gln

245 250 255

Ile Thr Tyr Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln Arg

260 265 270

Ser Ser His Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser Ala

275 280 285

Val Arg Ala Arg Gln Arg Thr Ile Val Thr Leu Pro Lys Leu Pro Glu

290 295 300

Leu Glu Val Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly Glu

305 310 315 320

Ala Arg Leu Asp Ser Val Thr Ile Asn Gly Asn Thr Ser Tyr Cys Val

325 330 335

Thr Lys Pro Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly Cys

340 345 350

Thr Met Asn Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala Val

355 360 365

Asn Asn Gly Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ser Gly

370 375 380

Ala Cys Glu Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu Leu

385 390 395 400

Arg Gln Arg Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr Gly

405 410 415

Thr Thr Ser Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp Val

420 425 430

Cys Thr Asp Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile Lys

435 440 445

Pro Ser Asp Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro Thr

450 455 460

Gly Glu Leu Tyr Ala Phe Lys Asn Ile Thr Thr Gly Lys Thr Leu Gln

465 470 475 480

Val Leu Pro Cys Glu Thr Pro Ser Gln Leu Ile Val Ile Asn Asn Thr

485 490 495

Val Val Gly Ala Ile Thr Ser Ser Asn Ser Thr Glu Asn Asn Arg Phe

500 505 510

Thr Thr Thr Ile Val Thr Pro Thr Phe Phe Tyr Ser Thr Asn Ala Thr

515 520 525

Thr Phe Asn Cys Thr Lys Pro Val Leu Ser Tyr Gly Pro Ile Ser Val

530 535 540

Cys Ser Asp Gly Ala Ile Val Gly Thr Ser Thr Leu Gln Asn Thr Arg

545 550 555 560

Pro Ser Ile Val Leu Leu Tyr Asp Gly Glu Val Glu Ile Pro Ser Ala

565 570 575

Phe Ser Leu Ser Val Gln Thr Glu Tyr Leu Gln Val Gln Ala Glu Gln

580 585 590

Val Ile Val Asp Cys Pro Gln Tyr Val Cys Asn Gly Asn Ser Arg Cys

595 600 605

Leu Gln Leu Leu Ala Gln Tyr Thr Ser Ala Cys Ser Asn Ile Glu Ala

610 615 620

Ala Leu His Ser Ser Ala Gln Leu Asp Ser Arg Glu Ile Ile Asn Met

625 630 635 640

Phe Gln Thr Ser Thr Gln Ser Leu Gln Leu Ala Asn Ile Thr Asn Phe

645 650 655

Lys Gly Asp Tyr Asn Phe Ser Ser Ile Leu Thr Thr Arg Ile Gly Gly

660 665 670

Arg Ser Ala Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Ser Gly

675 680 685

Leu Gly Thr Val Asp Gln Asp Tyr Lys Ser Cys Ser Arg Asp Met Ala

690 695 700

Ile Ala Asp Leu Val Cys Ser Gln Tyr Tyr Asn Gly Ile Met Val Leu

705 710 715 720

Pro Gly Val Val Asp Ala Glu Lys Met Ala Met Tyr Thr Gly Ser Leu

725 730 735

Thr Gly Ala Met Val Phe Gly Gly Leu Thr Ala Ala Ala Ala Ile Pro

740 745 750

Phe Ala Thr Ala Val Gln Ala Arg Leu Asn Tyr Val Ala Leu Gln Thr

755 760 765

Asn Val Leu Gln Glu Asn Gln Lys Ile Leu Ala Glu Ser Phe Asn Gln

770 775 780

Ala Val Gly Asn Ile Ser Leu Ala Leu Ser Ser Val Asn Asp Ala Ile

785 790 795 800

Gln Gln Thr Ser Glu Ala Leu Asn Thr Val Ala Ile Ala Ile Lys Lys

805 810 815

Ile Gln Thr Val Val Asn Gln Gln Gly Glu Ala Leu Ser His Leu Thr

820 825 830

Ala Gln Leu Ser Asn Asn Phe Gln Ala Ile Ser Thr Ser Ile Gln Asp

835 840 845

Ile Tyr Asn Arg Leu Glu Glu Val Glu Ala Asn Gln Gln Val Asp Arg

850 855 860

Leu Ile Thr Gly Arg Leu Ala Ala Leu Asn Ala Tyr Val Thr Gln Leu

865 870 875 880

Leu Asn Gln Met Ser Gln Ile Arg Gln Ser Arg Leu Leu Ala Gln Gln

885 890 895

Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ser Arg Tyr Gly Phe Cys

900 905 910

Gly Asn Gly Thr His Ile Phe Ser Leu Thr Gln Thr Ala Pro Asn Gly

915 920 925

Ile Phe Phe Met His Ala Val Leu Val Pro Asn Lys Phe Thr Arg Val

930 935 940

Asn Ala Ser Ala Gly Ile Cys Val Asp Asn Thr Arg Gly Tyr Ser Leu

945 950 955 960

Gln Pro Gln Leu Ile Leu Tyr Gln Phe Asn Asn Ser Trp Arg Val Thr

965 970 975

Pro Arg Asn Met Tyr Glu Pro Arg Leu Pro Arg Gln Ala Asp Phe Ile

980 985 990

Gln Leu Thr Asp Cys Ser Val Thr Phe Tyr Asn Thr Thr Ala Ala Asn

995 1000 1005

Leu Pro Asn Ile Ile Pro Asp Ile Ile Asp Val Asn Gln Thr Val

1010 1015 1020

Ser Asp Ile Ile Asp Asn Leu Pro Thr Ala Thr Pro Pro Gln Trp

1025 1030 1035

Asp Val Gly Ile Tyr Asn Asn Thr Ile Leu Asn Leu Thr Val Glu

1040 1045 1050

Ile Asn Asp Leu Gln Glu Arg Ser Lys Asn Leu Ser Gln Ile Ala

1055 1060 1065

Asp Arg Leu Gln Asn Tyr Ile Asp Asn Leu Asn Asn Thr Leu Val

1070 1075 1080

Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr Leu Lys Trp Pro

1085 1090 1095

Trp Tyr Ile Trp Leu Ala Ile Ala Leu Ala Leu Ile Ala Phe Val

1100 1105 1110

Thr Ile Leu Ile Thr Ile Phe Leu Cys Thr Gly Cys Cys Gly Gly

1115 1120 1125

Cys Phe Gly Cys Cys Gly Gly Cys Phe Gly Leu Phe Ser Lys Lys

1130 1135 1140

Lys Arg Tyr Thr Asp Asp Gln Pro Thr Pro Ser Phe Lys Phe Lys

1145 1150 1155

<210> 9

<211> 25498

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus BIV-Isolate 5.0327 Genome cDNA

<400> 9

ctttgagcac ctgaaaccaa gacgcgtcag tagtagggac tactggtgca gccatgatag 60

attggtgtcg gtagagaact agcgaagcta gggagataaa attatagact aatgctataa 120

tttttatctt tagtctataa ttttatctcc ctagcttcgc tagttctcta ccgacaccaa 180

tccaggtgcg tctgccacca agttggctac ccttcctagg ggcgctttcg cgcttgctca 240

ccattagatt acctggaaac cagccattca ggttggagtt tccccaggct cttttgtgtg 300

ggcattagcg gcttgtggtt tttgcacaaa atctaagcta cttaccgttc ctctgaccat 360

ccaccacttc tatagacagc actgattacc gtagggttta agtcacaccg gtctgcaccg 420

cccgtcagcg gacacattac ccagcatagc actccttgca ccgcgcctag gtaggataaa 480

accccctacc gggtgactct taaggcgttt cctccacggg atagccacta gtcactaggt 540

gtaagtgatc tgatctgggc gtattgtgtt gcgcaagtgt gatacccata ggagcgtgga 600

atcctattct gcggctcagt gcctgatata gctgtgaaat ggccaagaac aagtccaagc 660

gcgacgctat tgcgttgcct gaaaatgtac caccacctct gcaacttttc attcatgttg 720

cagctgctga agagggtcac cctaaggtta ctacttacct tggcaactat aacctctatg 780

ccaccaaggc tccgcctggc gtgcaggttc ttagtgctaa aacctctctt actgactttg 840

agaatgtctt tggagctcaa cccaccttgc gatcaattcg taatctggtt tgtgaggctc 900

gctcggctga atggacaact tccaagaatg cttttgcact caaagccact caacttgact 960

actctgatgc cgttttgagg gcaatgattc gtttctgccc tccaaaggtg tccacactcg 1020

ctgcctttgc tctttttggc cgattggtta aaattgagga caaggaactt gctgagttag 1080

ctcgtgatac tgcccttgag ttggcgtaca cggctaaaat tggtacatct cttgctgaca 1140

cgagatctgt ctcacttatt cataaggacg cttatctaac tctcagtaat gaggttgttg 1200

gcgtaacttt tactgccgca cttatggcaa aggctaccac tgttaatgga gcaatgcaat 1260

actcaaactt ttacctctac cctcgtgcca ctattaaggt aaccgatggt aaggctgaag 1320

caattgcaac taagcctctg tctgctgcca ctaaaggcaa gcaaatcaca gaggatgtca 1380

accttctccc tgactatcag cagctgcttg ttgatcaagt gactggcact gaggttaagg 1440

ttggagctct aacctatgtt aagaccactg attcgccacc cctttacttt cccaaagtca 1500

agggtggtgt tattggtatt gcacttaagc agcagggcac tgtggctaag aagctcaatg 1560

tagtcttcca tgctcaacct gatgatgttc tgctagcctt catacaactt cagcaattct 1620

tgaaccgtac ttcggattca agtgttgaaa ttactgattg ccagagttat gaagtatctc 1680

caactgtgac ggtcaaaatt ggcccgtcta aacctgggga tgtcatcgtg gctactgatg 1740

aggaatacct taaatgcttt gaaacccctg aggtaggtag gctctataag gttttccaaa 1800

ctcaatcttg ggctatcatt gagcgtgcct tctccagttt gaagatccgc gtgtccaaag 1860

ctttatcagc atttataagt tttctgcaaa accttgcaga taactttact gcaataagtg 1920

gtgttgtcac tgcactcatt cgtgaactcc aggatcttac cctggatgtg gcgacacgta 1980

tcactaacat acaatttgtt taccgtgccg gtaagcttat tgtcgacacg acaagtgtca 2040

tagctaaact tttccagcca ttttgtgatt ttatatcacc tttccttcgg aaagttgctg 2100

gttttgcaat ttacactgtt ggtaatcgca tgcttatgtt taccagcact ggcacctttc 2160

ttctcacaaa ggcaactact aagatactca ataaggcaaa gtacatcttt gatgtggagc 2220

ctgagtaccc agtagatgta acaacatcca aagttgtagt acatgaagca ctccagcaaa 2280

ccgacactaa gcctactaga gctctggagg ctgttgatgt cgttgttggt aatactgtac 2340

tgcaaatggc tactgatggc actgcgttct acccatcgga tggtacgcac gcctctcttc 2400

caggattcaa agcaggttcg gatgagcttt tcataagctt caactgcgac ctctttgatg 2460

atgagactaa tgctcaaatc aacgaaacac tcgctgcata tgagcttaac caactagtgg 2520

ctccaggtga ttctacaccg cgtcaaattg cgacgttggt tgtcgataca cttgcagatg 2580

ctataacaga ccactttccg gagaaaacca ttgatctacc tgaagactat caagtctttt 2640

ctgaccatga tgacctccca ctcgcacaat accacatccc tgatcacctg agcctgtata 2700

ttcaggctat ggaaggtgaa gatgatagtg gtgatgaaat atgtattgag gacgatgatt 2760

acgactgtcc tcaagccgac gaagacacag aaggagtaat tccccaacag tgggaacttc 2820

ctgatgttga taaattttta ctcaagatcc aggaacggaa gaccagcagc gacgaagtac 2880

ttagcgtcga cgtctatcct aaaccagagc cggtcggcaa tgttgggatt gacgacagcg 2940

cgtcggaaaa gaagccaaat ggggactcag taccggatcc tgaggtccat ccaacactag 3000

agagtgtgga tgttgaacga ccaaccgaaa cagcaaacca ggctgttgaa gacaaacctt 3060

ctgataccac ctttgtggtt gatgaggaac aattacaaga atcaacacca gaacatgaac 3120

tccgctccta tgaaggggag tttgattctg atgatgaaat tattattcct atagtaccag 3180

taacacctgc ggatttaaaa ccacagacta ttactataaa ggagtacttt aagtctgaaa 3240

aacttgagac tattaacgaa ggatccacag agtcagttac acaatctgac gattcgtttg 3300

acgagtcatt tgttgatgct gagtctgatg atccacaaga tcctgctgta tatgatgata 3360

caacaattat aacggacagc actgatgtag gcgatgagcc tgagacaact ctagctacca 3420

tcgttaacac acctctgaca ctcgataata acttgccacc tgaagccatt aaacaaccca 3480

gcccaactaa ggttgagtta gttgttggtg aattggcgag tattaaattt gacaattctg 3540

ttctagtcaa ccctgctaat gcgcaattaa caaatggcgg tggagctgct cgtgcaattg 3600

caaaattagc tggtccaaaa tatcaagagt actgtaatag tgtggctcct atctcaggac 3660

cgcttaccac ggactctttt gatgccaaga aatttggtgt agcctgcatc ttgcatgtag 3720

tgccacccaa aggttctgac cctaatgtac aagaactcct gtatcaagct tacaagagta 3780

tccttactga accagcacac tatgttatac ctatactagg tgctggtatc tttggatgca 3840

acccagtcca ctctctggat gcgttcagga aagcatgtcc aagtgacata ggtcgtgtca 3900

cccttgtcac tatgaacaaa aaccatttgc aggtgtggga tgctctcaat aggaccattg 3960

tacgcaccac tactgactat gatcaagtta ccaccaaggc ccttacaccc cagggagtgt 4020

tagaagccaa tctctttgat ggtgaggact ttgttcaaga accaaaaccc ggtcaaatct 4080

accttgaggt tactgaagaa gttcagaacc aagccaagga acttgacctt aaccttcagc 4140

aatactgcgt ctacctgaag acttgccacc ataaatgggt tgtgagtcgt acgaacgggt 4200

tgatgcatct aaaacaaaaa gataacaatt gttttgttag tgcaggtgta aacctgtttc 4260

aaaacactgc ttatcaactt agacctgcta ttgatgctct ctatagggag tatcttaatg 4320

gtaatccaaa tagatttgtt gcttggatct acgcatccac taaccgtcgt gttggtgaga 4380

tgggttgtcc acagcaagtt atttctttgc tcgttagtaa ctctgacgca gcattttcag 4440

caactacagc ctgttgtaac acctacttta accacacagg tgttatttca gtagctcgtg 4500

aatatgaccc aatacaacca aaggtctact gcatgaagtg tgatgtgtgg actcccttta 4560

caccccagag tggaaaaggt gcagttgcaa ttggtatttc tgcagatgaa cctaccggtc 4620

ctgccattaa atttgccgca gctcactgct ggtacactaa tggcaagaaa acagttaatg 4680

gctatgacac taaagctaat gttgtagcta cctatcatag gtttgacgtg cctaagcctc 4740

aacttgtcga ggacgtggtt gcgctgccta ctaaaaatga ctttgaagtt ctcaatgttg 4800

aagaactgcc gcaggatagt gtgctccatt tggacccacc tcctgtacag gccttacaac 4860

ctaaggctaa ccaacacatt gagattctag aaaacccaga ttatctggac attttggatc 4920

tttggattcg taaacccaaa ttcatcctcg taaagtcgtg gagtgttttg ggtagagcac 4980

tatgtaaggc aggtaaagtt gtctttgtca gtgcttcgct tttgacccgt ttctacaatt 5040

accttgtaga gattggtgct cttgactcaa caataaggtt gtcagtcgat cttacctgta 5100

aatttgttag aacggttctc ccatcgtcta acactgtaca caaaacttgt cttggtctgt 5160

attattcagc ccagacactt tttgtttctt tagcaccatt ccttatgtta ccagctgtag 5220

ttagtctgct taattcaggc tatacaattg gcacatattt gtatgcaaaa actggctggc 5280

cttgtaatta caatgccacg caacactttg attataattc ttactgtgca ggtgacttgg 5340

tttgtcaagc ctgttttgac ggtcaagact ccctacattt gtatccgcat ttacgtgtta 5400

atcagcagcc ccttcagacc actgactaca ctgtttatgc gctttcacta atactactat 5460

tagctaacat gactcttgtc atgggcacgc taatagttac tttctttgtg aacttctatg 5520

gtgtgcaaat accattttat ggtacacttt tgatagatta tcaatccgca ctggtgatta 5580

ctttctcagt gtactacttt tataaggtaa tgaagttttt ccgccatctc acacatggat 5640

gtaaaattcc aacgtgtgtg gtatgtgcca aacttcgtac cccacctact ataacagttg 5700

agactgtcgt tcagggcagg aaatacccat ctgttattga aacaaatggc gggtttacaa 5760

tttgtaaaga acacaacttc tattgcaagg actgctcttt acaaacaccc ggcactttca 5820

tcccgacaga agctattgag tcgctctcac gagctaccag gcttagtgtc aaaccaacag 5880

caccagcatt cttacttgct agagatgttg agtgccaaac tgatgttgtc gttgctcgcg 5940

caatgcataa ccaaaatgcg catgtgtgca tttcaaaata ctctgatatc cgtaccgttg 6000

accaactact taagcctact ccactgtttt catacactcc cgatgttatc atcgcggcag 6060

actttgacaa cagaggtagt cttaagacag ctaaagaatt agctgtggtt ttgtcaatgg 6120

accttaaacg tactataatt atcattgatc aggcctattc tagacctatt gataattatc 6180

aggaagttgc ttctcgtatt gagaagtatt acccagttgc aaagatcaca cccacaggtg 6240

acatctttac agacattaag caagcgacca atggccaagc tagtgactct gctattaatg 6300

cagctgttct ggctgtccag cgcggtcttg attttacaat tgacaaccct aacaacatat 6360

taccacatta cgcctttgac ttttcaaccc tcaatgcaga agaccagtct accattttgg 6420

agagtggttg tgctaaaggc aatctcaagg gcactaatgt tggtgttgtt ctttcagcta 6480

gccttgttac acgtcttagt cagcaggcta tacgtgtgat tgctaatgct gcttcacgta 6540

atggtgttac atgcgctgtt actccttcta cacttgttat gcgtgggaat attgcaacac 6600

agcccttgac tcgcatcaaa gctggtgcac ctcccatgcg tcaaaaaatt ttatgtgtta 6660

tcctggcact tgctattgtg tactttgctg ctatggcttt tggctttttg gcaagtcaaa 6720

ttacgcttaa tacagtgcct acgattaaat ctgatatccg cgcctctacc ttctacgttg 6780

ttagagatgg agtcttggat actgttcgtt caaatgacaa gtgctttgca aataagtttt 6840

tggcatttga tagcttcatt caagcacctt acactaattc acctgactgt ccagttgttg 6900

tgggagttgt tgatgtaacg acgcactcta ttcctggaat tccagcaggt gtcattcata 6960

gagacggtct catacttaac atttatgaac agtctcttta tgaaactcat cagcgtcagt 7020

ctatggttag ggatgcgttg tcactcaaga cagcaaatct ctttaaccta ggcaagcgtg 7080

ttgtagtagg atacactcaa catgaagttg ttgtgggtac ctcctatttt aattctcctg 7140

cactttttaa tgcaaagtgc accttcttac agtatcagga cactagacaa ctctattgct 7200

atgatactgt tcctactgaa cataagctct actctgatgt gcttccgcac gtcgagtata 7260

aggctattga cattaatggt gatcttgttc ctttcaagat accagagcag ataatgttct 7320

atccacatat tgtgcgctat actagcaatt cctattgccg tatggggcat tgttttaata 7380

ctaaccctgg tatttgcatt tcatttacgg acgaatttcc gtatagtgaa aatgtcaaac 7440

ctggtgtgta ctgtgctgat acctctttgc agttgttttc aaacctcgtt ttgggcactg 7500

tatctggtat tcacatcttt acatcaacag ctgcattgct tggatctact attgtgatca 7560

tactatgcgt tgttgctgtt cttgcagttc agcgattctt caaggagtac acaacttttg 7620

ttatgtacac ttgtggtctt gctcttgtca acattgtagg cattgcactt atgtacaagt 7680

gccttgtctt cgcgattttc tattatgcaa tctaccttta ctttgtcctt actttcccct 7740

cctttaagag gaatgtggca ttgttttact tcgctgtagt gatcgtgccg cacgtgagta 7800

acatgcaatt gcttgcgctc attgtgtgta gcattatcta ctttctctac acctatgttc 7860

atactgtagc taagacagct gggaaatttt cttccttctt agacgcagct aaagctactt 7920

ttgtcattga caatgaaaag tacgtgttgc ttaaagacct cgctggtgct gaatttgacc 7980

agtatctggc ctcttacaac aagtacaaat atttttctgg tactgcttct gataaggatt 8040

atgataaggt ctgtatggca tttcttgcca aggctttgtc atcttttcgt gaaggaggcg 8100

gttcacagtt gtacacacca cctaaatttg cagttgttca gagtcttaag accaagctgc 8160

aagcaggtat caaaatcctc ctgcaccctt caggtgtagt tgagcgatgt atggtctcag 8220

ttgtctacaa tggatctgca ttgaatggca tctggcttaa gaatgttgtc tactgcccac 8280

gccatgtaat tggaaaattc cgtggtgacc agtggactca catggtctca attgctgatt 8340

gccgcgactt tatagtcaag tgtccaatac agggtattca gctaaatgtc caatcagtta 8400

agatggtagg agctctcctc cagttaactg ttcataccaa caacacagcc actccagact 8460

ataagtttga aaggctacaa ccaggatcat cgatgacaat tgcttgtgct tatgatggca 8520

ttgtacggca tgtctatcac gtggtcctcc aacttaataa tcttatttat gcaagcttcc 8580

ttaacggagc ttgtggtagt gtgggttaca ctcttaaggg taaaacactc tacttacatt 8640

acatgcacca cattgagttt aacaacaaaa ctcatagtgg tacagatctt gaaggtaact 8700

tctatggccc ctatgtggat gaggaagtta ttcagcaaca aacagcattc cagtattaca 8760

ctgataatgt tgttgctcaa ttatatgcac acttactgac tgttgatgct agaccaaaat 8820

ggctggcaca atctcagata agtatcgagg atttcaactc atgggctgct aacaattcct 8880

ttgctaactt cccatgtgaa caaactaata tgtcctacat tatgggactc tcgcaaacag 8940

ctcgagtccc tgtagaacgt atcctcaata ccattataca gctaaccacc aatagagatg 9000

gtgcttgtat tatgggatct tatgatttcg agtgcgattg gacgccagag atggtataca 9060

atcaggctcc aatttcattg cagtcaggag tagttaagaa aacttgtacg tggttcttcc 9120

acttcttgtt tatggctatt accatgctac tcgctgccat gcatgttttc cctgtacact 9180

tgtatccaat agtactgcca tgcttcactg tcgtggcatt cctgttgact ttaaccatta 9240

aacacactgt tgtgtttacc actacatact tgcttccgtc acttttgatg atggttgtaa 9300

atgctaacac tttttggata ccgaacacat ttctgcgcac ctgctacgaa actatattcg 9360

gttccccaat tgctcagcga ctgtatggtt acactgttgc tctttatatg ctgatctatg 9420

ctggacttgc aatcaactat acgttgaaaa cactccggta tagagcaact tcattcttat 9480

ctttttgcat gcagtggttt caatatggtt atgttgcaca cattgcgtac aaactgctta 9540

ataaaccctg gacagaatca ctactcttca cagccttcac aatgctaacc agtcatccct 9600

tgttggctgc tcttagctgg tggctagctg gtcgcgtaac tctgcccatt atcatgcctg 9660

acttagctat tcgtgttttg gcgtataacg tcattggcta tgtcatatgt gttcgatttg 9720

gccttatgtg gcttgcaaat cggttcacaa ctgtacctat gggcacatac cagtatatgg 9780

tgtctgtaga gcaacttaag tacatgatgg cagttaagat gtccccaccg cgtaatgcgt 9840

ttgaggtgct tatagccaac attagacttc ttggtttggg tggaaaccgt aacattgctg 9900

tttctactgt ccaaaacaaa attcttgatg caaaagctac tgctgttgtt gttgctaacc 9960

ttcttgaaaa ggctggcgtc acaaacaagc acgctatttg caaaaagatt gtgaaactcc 10020

acaatgatac ccttaaagcc accacttatg aggaggttga ggtagcactt gtgaaacttc 10080

tttctcacat aattgagttc ttgccaactg atcaggtaga tgcttatcta gctgatgcgg 10140

ccaatgctca acatgttaat acctatttag acaacttgct tgagaacaaa gctgttgttc 10200

aggctgttgc cgatatcaac attaatctgg attcttatag aatttataag gaggcagatg 10260

ctatttataa acgatctgtt gagatgaacg aatctccgca ggagcaaaag aaaaagctta 10320

aagctgttaa cattgcaaag gcggaatggg agcgtgaggc tgcttctcag cgtaagcttg 10380

aaaagcttgc tgatgctgct atgaagtcta tgtatcttgc agaacgtgct gaggatcgtc 10440

gcattaagct aacctctgga cttactgcaa tgctttacca tatgcttaga cgtcttgact 10500

cagatagggt aaaagctctg tttgagtgcg ctaaggcaca aatcttgcca atacatgctg 10560

tagtcggaat ttctaatgac aaccttaaag ttatttttaa cgataaggac agctactctc 10620

attatgtaga gggcaacaca cttatacata agggagttcg ctacactatt gtgaagaaac 10680

tctccttaga taatgcacct attgaaggcg taccagaaga attccctgtg gtcgttgaga 10740

ctgttaggga aggtgtgccc cagttgcaaa ataatgagct atgtttgcgc aatgttttca 10800

ctgctcagaa cacagctcag gacttcaatg gcaatgaatc cactgtaaaa tctttttatg 10860

ttactagaac cggtaagaag attttggttg ccattacatc aactaaagac aatcttaaga 10920

ctgtgacctg ccttactgag accggtaaga cagtccttaa cttggacccc cctatgcgct 10980

tcacacatac cgtaggtgga aaacagtctg ttgtctatct ctattttatt cagaatatta 11040

gttcactcaa cagaggtatg gttattggcc acatctctga aactactatc cttcaggcaa 11100

gtggcactca aattgagtac cagcaaaatg cctctctttt gacctatttg gctttcgctg 11160

tagaccctaa gacagcctac cttaagcatc ttgctgatgg tgggtctcct atacagggtt 11220

gtattcagat gattgctact atgggtcctg gatttgcagt tactactaaa ccacaaccta 11280

atgagcatca gtattcttat ggtggtgctt caatttgtct ttattgccgt gctcatatac 11340

cacatcctgg tgttgatgga cggtgcccct acaaaggccg ctttgttcac atcgacaaag 11400

ataaggaacc tgtttccttc gctttgactc atgagccatg cagttcttgt caacggtggg 11460

ttaattatga ctgcacctgc ggatctagtc tgcagaattc ggcttattta aacgcgtaac 11520

gggttctagt gacgcccggc tagaacccct gcagcctgga actcaaccag atgctgtaaa 11580

aagggccttc catgtgcata atgataccac ctctggtata ttcttaagca caaaatctaa 11640

ctgcgctcgg tttaaaacca cacgcagtgc cctgccttta cctaataagg gagaggttga 11700

attgtacttt gttactaagc agtgtgcagc taaagtcttc gaaatcgagg aggaatgcta 11760

caacgctctt agtacagagc tttatactac tgatgataca tttggtgtcc ttgccaaaac 11820

tgagttcttt aagtttgaca agatacctaa tgtcaatcgc cagtatctga ctaaatatac 11880

actcctggac ttggcttatg ctctacgtca tttgtcaaca tctaaggatg ttattcaaga 11940

aatcttgatc accatgtgcg gaacccctga agattggttt ggggaaaatt ggtttgatcc 12000

aattgagaac ccatcctttt acaaggagtt ccataaactt ggggatattc ttaaccgttg 12060

tgttcttaat gccaataagt ttgctagtgc ctgtatagac gctggtcttg ttggcatatt 12120

aacacccgac aaccaagacc tcctgggtca gatctatgac tttggagatt ttattattac 12180

acaaccaggt aatggatgtg tggacttagc atcctattat tcttatttaa tgcccattat 12240

gtccatgact cacatgttaa agtgtgagtg tatggatagt gatggcaacc cacttgagta 12300

tgatggattt cagtatgact tcacggactt caagcttggc ttgttcgaga agtattttaa 12360

gtactgggac cgtccttacc atcctaacac tgttgaatgt ccagatgacc gttgcgtatt 12420

gcactgtgcg aacttcaatg tgttgtttgc tatgtgtata cctaatacgg catttggcaa 12480

tctttgttca agagctactg ttgatggcca ccttgtggtc cagacagtgg gtgtacactt 12540

gaaagaacty ggtatagtcc ttaaccagga cgttaccaca cacatggcaa atattaatct 12600

aaacactcta ttgcgattgg ttggtgatcc caccactatt gcaagtgtct cagacaagtg 12660

tgtagattta agaactcctt gtcagacctt ggctactatg tctagcggaa ttgctaaaca 12720

gtcagtcaag cccgggcatt ttaatcaaca cttctacaag catttgcttg atagtaacct 12780

attagaccaa cttggaatag acattcgcca cttctactat atgcaggatg gtgaagcggc 12840

tatcacagac tacagctact acaggtataa tacccccacg atggtagata tcaagatgtt 12900

cttattttgc cttgaggtgg cagataagta tcttgagccc tacgaaggtg gatgtattaa 12960

tgcacagtca gttgtggtct ctaatttgga caagtcagcg ggctacccct ttaacaagct 13020

aggtaaggct cgtaactatt acgacatgac tcatgccgag caaaatcaac tgtttgagta 13080

tacaaaacgc aatgttttgc ctacactcac tcagatgaac cttaagtatg caatttcagc 13140

caaggatcgt gctcgcactg tggcaggagt gtctataatt agcaccatga ctaacaggca 13200

gtaccatcaa aagatgctga aatctatttc acttgcacgc aatcagacca tcgtgattgg 13260

aacaaccaaa ttctatggtg gttgggacaa catgttacga cgactgatgt gtaatatcaa 13320

caatcccatt ttagtgggtt gggattaccc taagtgtgat cgttctatgc caaacatgct 13380

gcgcattgcc gcttcgtgct tgctagcacg aaaacacact tgctgtaacc aaagccagcg 13440

attctaccgt ttggctaatg aatgttgcca agtactatct gaagtggtag tctctggtaa 13500

caacctctat gtaaaaccag gtggcactag cagtggtgat gcaaccacag cttatgccaa 13560

ctcggtattt aacatcttac aggtggtttc tgctaatgta gccaccttct tatcaacttc 13620

caccacgaca catcttaata aggacattgc ggacttgcat cgtagtcttt atgaagatat 13680

ttatcgtggt gactctaatg atatcaccgt catcaataga ttctaccagc atctccaaag 13740

ttactttgga cttatgatat tgtctgatga tggtgtcgca tgcatagact cagccgttgc 13800

aaaggctgga gctgttgctg atcttgatgg tttccgagac attttgtttt accaaaacaa 13860

tgtttacatg gcagactcaa agtgttggac agaaactgac atgaatgttg gccctcatga 13920

attttgctca cagcatactg tgttagcaga gcatgatggt aaaccttact acttacctta 13980

cccagatgtc tctcgcattc tgggtgcatg catctttgtg gatgacgtta acaaggctga 14040

ccctgttcag aaccttgaac gttacatctc acttgcaatt gatgcatatc ccctcaccaa 14100

ggttgaccct attaagggta aagtctttta tttgttacta gactacatac gtgttcttgc 14160

tcaggagtta caggacggta tccttgatgc tttccaatca ctcactgaca tgtcgtatgt 14220

aaataacttt atgaatgagg ccttttatgc tcagatgtat gagcaaagtc ctacactaca 14280

ggccagcggt gtttgtgtgg tgtgtaattc acccactata ctgcgctgtg gtgattgcat 14340

tcgtcgacca ctactttgtt gcgtctgtgc ctaccagcat gttacgcaga ctacacataa 14400

acgtatcatt gctatcaaca actacatctg tagtgttgag aattgcaatg aggacaatgt 14460

tgaaaaactt ttcatttctg gcactgcgat ttattgtgag aatcacaaac ccacgctgtg 14520

catacccatt gtagctaatg gttctgtttt tggtatctat cgccacactg cccgtggtag 14580

tgatgacata gacctcttta acgagcttgc tacatctaac tatgacacta ttgaacctta 14640

tcagaaggcc aatcgtgcac ctttatcact tatgctcttc gctgctgaaa ccattaaggc 14700

actcgaggag tctatcaaga agtcatatgc taccgcaacc gtcaaggatg tgtatgacca 14760

acgcttcatt aaacttctat gggaacaggg taaaaagccg ccacccataa cgaagaacca 14820

cattttcact ggctaccatt ttaacaagaa tggaaaaacc caagttggtg attacattct 14880

tgctaaaaca gatggcagtg acacttatac ttacagagga acatctacct acaaactcca 14940

aacaggtgat gttctagtct taatggcaca tgttgttaca ccgctctcag caccccctgt 15000

gttaacgcag acaacatatg tcagaaaatc acttttaccc gactctgttg gtgcgtctta 15060

ttatgtgcaa cattttaagt catataatga gatagctatg cagagggtta caacagtatt 15120

aggtccacca ggcacaggta agtcaacctt tgctattggt ttggctaagt actttcccag 15180

tgcacgtatt tgctacactg cgtcttcgca tgcagcaatc gatgcactct gtgaaaaagc 15240

tttcaagaca atacctgtag gccaatgcag tcgtatcgta cccacacgta caactgttga 15300

gtgctttcag gagtttgtcg taaataacac aactgcacag tatatcttct cgactatcaa 15360

tgccttacct gacattaagt gtgacattgt agttgtagat gaggtttcta tgttgaccaa 15420

ttatgagctt tcctctgtga atgctcgttt ggtttacaat cacattgtgt atgttggtga 15480

tccttatcag ttaccttcac ctagaactat gcttacgtct ggccagcttt cgccagctga 15540

ctataacgta gttactgata taatggtaca tgcaggagcg gatgttatgc tcgacatgtg 15600

ctacagatgc ccacgtgaaa tcgttgagac agtgtctaaa cttgtctacg ataacaaact 15660

aaaagcggcg aaaccgaact caagacagtg ttacaagacc attgtgaact ttggtcctgg 15720

agacgttgct catgagggac aatctgccta caacgaagca cagttgcgtt tcgcactcgc 15780

atttagacaa caaaagcggt gggataacgt gactttcata tctccatata atgctatgaa 15840

tgtgaaagca tccttagcag gtttctctac tcagaccgtt gactcttctc aaggttctga 15900

gtatgattat gttatctttt gcgtgaccac tgattcagca cacgcactta acatggctcg 15960

tttgaacgtt gcccttacac gcgcaaagat aggtatcctt gtggtgttta ggcaggcaaa 16020

cgaactttac aatagtttgc agtttgaatc tattgattca cagcttcagt cgagtgctga 16080

gaaaaacctc acaccactgt ttaagcgctg cggctatgag tataatggcg tccatccagc 16140

tcatgctttg acctggcatg attgtggtgc agagtaccgc tgtgaggagc cacttgctaa 16200

attagtagga gttgccgatg gcactcttat atcatacaaa accctagtat ccacacttgg 16260

gtttcttcca tcacttaaaa ttgatgcata tcataatatg ttcctaacac gtgacgcgtg 16320

tcgcacctat gttcagagtt ggatcggcat agatgttgaa gcagcacacg ccataaaacc 16380

taacaccggg actaacctgc cattgcaaat aggttttagt accggaaaga atttttcagt 16440

cactccagag ggaatttggg taaacgagca cggatcttgc actgagcccg tccctgccaa 16500

aatacctcct ggagaacaat ttcgtcacct taaaaaggac atgcgccagg cgcgtccttg 16560

gaaggttgtt cgacgtgaga ttgctactca cattgctgag gtagctcctc acactgatta 16620

tatatgcttt gtcacttggg ctcaccagct tgagctagcg acaatgcgct actttgtcaa 16680

actaggtatg gaagagaaat gcttttgtgg caggcgggct tgtttcacta atggaactga 16740

gttcgcttgc aaagcacacc attctctcac cattccacaa tgtgattatg tgtacaatcc 16800

attcctcatc gacgtggcta cgtggggatt ctcgggacgg ctttccacca accatgacgc 16860

tgtgtgcaca tatcatgcta atgcccatgt tgcatcagct gatgcaatca tgacggtatg 16920

tttagctatc catgaactgt tcagtactgt tgactggaac tttgaatttc cagtaactgc 16980

tgagcaatcg caacttaaca aggcctgtcg cttagtacag gcgaattact taaatatact 17040

actcactaca accaaagcca cggtggttca cgatattggt aacccaaaag gtatccctat 17100

cgtgcgcaaa cctggtgtta aatatcactt ctatgatcaa gcacccattg tcaaacacgt 17160

tcaaaaacta aagtacaagc cagagatgga ggcccgtttc accgatggtt tgactatgtt 17220

ttggaattgt aatgttgaca cataccctgc taacgccctt gtgtgccgct acgacactca 17280

tcggcagaag catttaattg gacctaatgg ttcagcacta tatgttaata agcatgcttt 17340

tctcacccct gagatgcata cttatgctac acataaactc aacttggctc cactcatcta 17400

ctactccacc acagattgta gtagtgaaca gcctattgtt gttacctaca gagattgtgt 17460

cacccggtgc aatactggaa aaactctctg tccaaatcat gctcttgaat accaagagtt 17520

tatcaatgca tacaatctca tggctcgcca tggatttaat gtttacatac cacgcaatgt 17580

caacgtttac aactgttggc ttactttcac taatctccaa aaccttgaaa acttagctta 17640

caactgttat tataagaact gcaatgctca cgttgatggg cagcttgatg tagttattaa 17700

taataacgct gtatatgcta aggtcgacaa taatcttgtc aaacttttcg acaaccgcac 17760

taacttacct gtctcagtgg cctttgaaca ttacactaac aggcataccc gttcactgcc 17820

aactacacag ctgttatctg gtttaggcgt aaccgccacc agaaatttca ctgtgtggtt 17880

cgacaatgat acaattttcc aatacactat taatgtatct acgtatactg acatcgaccc 17940

tagtacccat gttgtcctct gtgatgatag gtacggaaca gattggagtc agtttaacca 18000

acttcctaat gcagtattcc tcaccaaaac taaggtgaag aaaacagaac cgtttgtttg 18060

tacagcactg accctaaatg gcgtcgccat tgacggtgaa gagctataca tctatgtacg 18120

ctataacaat caactgacca catttgctac tacttgtaca cagggtagaa atgttgagca 18180

gtttatacct aaaacaccta tggaaagaga cttccttgag atgtctcaac agtccttcat 18240

ccagcaacat caattgcagg aactgggtgt tgaacacatt atctatggtg atgattccag 18300

tccagtcatt ggcggaactc acacacttat ctcactagtt aaaaacaagt ttgaacatca 18360

gcttgtcaac catgtttaca acccagtcca gaactgtgtt gttacctcac ctaacgcaag 18420

ctccaagaac gtttgcactg ttcttgatgt tcttcttgat gactacattg acatcataag 18480

acaagcacat gccagttaca caagtaaatc caaagtattc actgtgtcaa ttgacaacca 18540

acaaattaga ttcatgcttt ggcatgatga gcaagtcaag acttgctacc caatcttaca 18600

gtcacttacc aatggttacc agatgccatc tgtgtacaaa acattggtta ctgacttaca 18660

accagctgac atccctaatt atcattccta caccccccgg gtgcctggag tagttaagaa 18720

tgttatcaag taccgccaac ttttcaacta catagttaaa aaggataggt tggcagtacc 18780

acacaatatg accgtattac accttggagc tgcatctgca ctaggtacag caccaggttc 18840

ttcagtcata aaacaaatgt ttcctgaagg aactgttctt attgacctcg atataagaga 18900

gttcacttca gatgctaacc aaataatagt tacagactac agaacttaca taccaccaca 18960

ccacgtagac gtcatatttt ctgacctcta ctgttgtgat gacatacact tctttgacaa 19020

tctaataagg atagttaagg agaggctcgc cctcggtggt tctatctttg ttaagataac 19080

tgaacattca ttctcacccg aactctactc acttgcgggt tggttcgatg attatcaact 19140

attttgcaca gcagtcaatg cctcgtcttc agaagcattt ttatgctgtt ttaattattt 19200

ggggcttgct aaggaaaaca ttaatggttt taacttacat gcttcctata ttcaatggcg 19260

caatgaaata gcgttgacac caacctattc tcctttagcg gacaacccgg ctacggcctg 19320

taagctaaaa gcaacgccta ttatctcggc tcgtgagtta gagaagaagc ctattcttcg 19380

ctatctcgtt gcatcagggc gccttcttgt gaggccacca gaatgcagag agctctattg 19440

attatgacct tattttgtct cgttcgagca aagtttgctg atgatctact cgatttgctc 19500

accttcccgg gtgcacatcg cttcttacat aaacccacga ggaattccag cagtctctac 19560

tcgcgggcta ataataattt tgatgttggc gttcttcctg gctaccccac taagaacgtt 19620

aacctcttct caccacttac taactccact ttgcccatta atggccttca tcggagttac 19680

caaccactca tgctgaattg tcttactaaa ataactaacc acactctcag catgtatctc 19740

ctacctagtg agatacaaac ttatagctgc ggcggtgcca tggttaaata ccagacacat 19800

gatgcagttc gtatcatttt agacctcact gccactgacc acatctctgt tgaagtcgtt 19860

ggccaacatg gtgaaaatta tgtgtttgtt tgcagtgagc agtttaacta caccactgca 19920

ttacacaact ctaccttctt ctcacttaat tctgagcttt attgctttac taataacacc 19980

tacttaggta ttcttccacc tgatttaact gactttacgg tctaccgtac tggtcagttc 20040

tatgctaatg gttacctttt aggtacttta cctattacgg ttaactatgt taggttgtat 20100

cggggtcatt ttagtgccca ctttgccctt gcaaacctaa ccgatacact cataacactt 20160

accaatacta ctatatcgca aatcacttat tgtgataagt ctgtagttga ttcaatagca 20220

tgccagcgct cttctcacga agtggaggat gggttttact ccgaccctaa atctgccgtt 20280

agagctaggc aacgtactat tgttacacta cctaagctcc ctgagcttga agtagtgcag 20340

ttaaatattt ctgcacacat ggattttggc gaagccagac ttgacagcgt taccatcaat 20400

ggtaacacat cctattgtgt cactaagcct tacttcaggc ttgaaactaa ctttatgtgt 20460

acaggttgca ctatgaatct gcgcactgat acctgtagtt ttgacctgtc agcagtaaac 20520

aatggcatgt cattctctca attctgtcta agcactgaat ctggtgcttg tgagatgaaa 20580

attattgtta cctacgtatg gaattacttg ctaaggcagc gtttgtatgt tactgctgta 20640

gagggccaga ctcacactgg aaccacttca gtacatgcaa cagacacttc tagtgtaatc 20700

actgatgtct gcactgacta cactatctat ggagtctctg gtactggcat tattaagcca 20760

tcagatctct tattgcacaa tggcatagca ttcacctctc caacaggtga gctttatgca 20820

tttaaaaata taaccactgg caaaaccctt caggtcttac cgtgtgaaac cccttctcaa 20880

ctgattgtga taaacaacac cgttgtcggt gctatcacat ccagtaattc aactgaaaat 20940

aataggttta ctactactat tgtcacacct actttctttt attccacaaa tgccaccact 21000

ttcaactgca ctaagcctgt tttgtcctat ggacctatca gcgtgtgtag tgatggtgca 21060

attgtgggaa catccacatt acagaatact cgaccatcca tagtttcact atacgatggc 21120

gaagttgaaa taccatctgc attttctctt tccgttcaga cggagtactt gcaagttcaa 21180

gcagagcaag ttatagttga ttgtcctcag tatgtatgca atggcaacag ccgttgtcta 21240

caattactgg cacaatacac ctcagcttgc tctaacattg aagcagctct gcattcctct 21300

gcacagttgg atagcagaga gattataaat atgtttcaaa catcaacaca gtccttgcag 21360

ttagctaata ttaccaactt caagggtgac tacaatttta gcagcatact aaccaccaga 21420

attggtggca gatctgctat tgaagacctt ctttttaata aagttgttac tagtggcctt 21480

ggcactgttg atcaggacta caaatcctgc tctagagaca tggccatcgc tgacttagtt 21540

tgttcccagt attacaatgg catcatggtt ctacctggtg ttgttgatgc tgagaaaatg 21600

gcaatgtata ctggctctct tactggagct atggtatttg gaggactgac tgccgcagcg 21660

gcaataccat ttgccacggc agtacaagct cgcctcaatt atgtcgcact gcaaacaaat 21720

gtactacaag aaaaccagaa aattcttgca gaatcattta accaagcagt tggcaatata 21780

tcacttgcac tatcttctgt taatgatgcc atccagcaaa cttctgaggc tcttaacacc 21840

gtagctattg ctattaaaaa gattcaaaca gttgttaacc agcagggcga ggcattatca 21900

cacctgactg cacagctgtc aaacaatttt caagcaattt cgacttctat tcaagacatt 21960

tacaaccgtc ttgaggaagt agaggctaac cagcaagttg accgtctcat cacaggacgg 22020

ttggctgcac ttaatgcata tgttactcag ttactcaatc agatgtctca gattagacaa 22080

tctcgattgt tagctcagca aaagattaat gagtgtgtca aatcacagtc gtccagatac 22140

ggtttctgtg gaaatggcac acacatcttc tcacttacac agactgcacc aaatggcata 22200

tttttcatgc atgcagtgct agtacccaac aaattcacac gtgtcaacgc ttctgccggc 22260

atttgtgtgg ataatacgag aggctactca ttgcagcctc aacttatact ctaccagttt 22320

aataactcct ggagagttac acctagaaat atgtatgaac ccagactgcc ccggcaggct 22380

gatttcatac aattaactga ttgcagcgtt actttttaca acaccaccgc tgctaatctt 22440

cccaatatta tccctgacat tatagatgtc aatcaaacag tcagtgatat tattgacaat 22500

ttacctacag caacacctcc tcagtgggat gttggtatct ataacaacac tattctcaac 22560

ctcaccgttg agattaatga tctacaagag cggtctaaaa acctctcaca gattgcagat 22620

cgtttacaaa attatattga caatcttaac aatactctag ttgaccttga atggctcaac 22680

agagtggaaa cttaccttaa atggccgtgg tatatatggc ttgccattgc cctggctctt 22740

attgcatttg tgacaatcct cataacaatc tttctttgta ctggttgttg tggtggttgc 22800

tttggttgtt gtggcggttg ttttggcctt ttctctaaga agaaaaggta taccgacgac 22860

caaccaacac cgtcctttaa gtttaaggaa tggtagtcga cgactgggcc gttaccatcc 22920

ctggacaata tattattgct atactagttg tcatctgcat tggtgtggca ctacttttta 22980

ttaatacttg cttagcttgt gttaaattat tttacaagtg ctacctaggg gcagcatatc 23040

ttgttaggcc tattatagtg tactactcca agccgaaccc cgtacctgag gatgagtttg 23100

taaaagtaca ccaatttcct agaaacactc actatgtctg acgcagaaga gtggcaaatt 23160

attgttttca ttgcgatcat atgggcgctt ggcgtcatcc tccagggagg ctatgccacg 23220

cgtaatcgtg tgatctatgt tattaaactt attctgcttt ggctgctcca acccttcacc 23280

ctagtggtga ccatttggac cgcagtcgac agatcatcta agaaggacgc agttttcatt 23340

gtgtccataa tttttgccgt actgaccttc atatcctggg ccaagtactg gtatgactca 23400

attcgtttat taatgaaaac cagatctgca tgggcactct cacctgagag tagactcctt 23460

gcagggatta tggatccaat gggtacatgg aggtgcattc ccatcgacca catggctcca 23520

attctcacac cagtcgttaa gcatggcaag ctcaagctac atgggcaaga gctggccaat 23580

ggcatatcag tcagaaatcc gccacaggat atggtgatag tgtcaccaag tgacaccttt 23640

cactacactt ttaagaaacc tgtggaatca aacaacgatc cagaatttgc tgttctgata 23700

taccagggtg accgcgcttc aaacgctgga cttcacacca taaccacttc aaaggccggt 23760

gacgctcgcc tgtataagta tatgtaatgt gcaactgcca tctgcagctg cgagatttat 23820

atagattgtg caataagcgg cacatcagaa gagaggatgt tcctgagctt attgaccctc 23880

tcgttaaaac tcgctgtttt gcttacagtc tcgtggttct tgctaatgct aatccaattg 23940

catttagcat actacctcgg aaaattctta tcaatggtga gcctttactg cttgaatatg 24000

gtagcatata tggtaaagac tttatcattc gaccatcgct ccaagtcatt cttgaagatg 24060

aattaaatta aagttttgac accaatctat catggctgca ccagtagtcc ctactactga 24120

cgcgtcttgg tttcaggtgc tcaaagctca aaacaaaaag gctactcatc ctcagtttcg 24180

tggcaatgga gttccgctta actccgccat caaacccgtt gaaaaccatg gctactggct 24240

gcgttacacc agacaaaagc caggtggtac tcccattcct ccatcctatg ccttttatta 24300

tactggcaca ggtcccagag gaaatcttaa gtatggtgaa ctccctccta atgatacccc 24360

agcaaccact cgtgttactt gggttaaggg ttcgggagct gacacttcta ttaaacctca 24420

tgttgccaaa cgcaacccca acaatcctaa acatcagctg ctacctctcc gattcccaac 24480

cggagatggc ccagctcaag gtttcagagt tgaccccttc aacgctagag gaagacctca 24540

ggagcgtgga agtggcccaa gatctcaatc tgttaactcc agaggcacag gcaatcagcc 24600

caggaaacgc gaccaatctg caccagctgc ggtacgtcgt aagacccagc atcaagctcc 24660

caagcggact ttacccaagg gtaaaaccat ttctcaggta tttggcaacc ggtctcgtac 24720

tggtgccaat gtcggctctg cagacactga gaagacgggt atggctgatc ctcgcatcat 24780

ggctctagcc agacatgtgc ctggtgttca ggaaatgctt tttgctggcc accttgagag 24840

caactttcag gcgggggcaa taacccttac cttctcctac tcaatcacag tcaaggaggg 24900

ttctcctgac tatgagagac ttaaggatgc gctcaatacg gtcgttaacc agacctatga 24960

gccacctacc aaaccaacta aggacaagaa gcctgacaaa caagaccagt ctgctaaacc 25020

caaacagcag aagaaaccta aaaaggtaac tctgccagca gacaaacagg attgggagtg 25080

ggatgatgct tttgagataa agcaggaatc agcagcgtag acatcaatct atgtctgtta 25140

aacccaccca actccactca aatatctctt tggttccaga gagtcgtagt gtatagccag 25200

agagccagtc agagggcgct atcatgcaaa ctagggctgg ctactctagc acagaatcac 25260

atcccgataa tcaacagtgc tagaaggttg attataccat ttaatatgcc gaggccacgc 25320

ggagtacgat cgagggtaca gcataatctc aacttttgtt gagccacaat tttaatccta 25380

attggagaag gccaaaggac tgtactactt ttgtgggtgt agcagtcgcc cagtgggaaa 25440

gcgccaacta ggttacaatt gtggtgggga caaattaggg gaaattaaat tggcttat 25498

<210> 10

<211> 25498

<212> RNA

<213> Porcine Deltacorona Virus ssRNA +

<400> 10

cuuugagcac cugaaaccaa gacgcgucag uaguagggac uacuggugca gccaugauag 60

auuggugucg guagagaacu agcgaagcua gggagauaaa auuauagacu aaugcuauaa 120

uuuuuaucuu uagucuauaa uuuuaucucc cuagcuucgc uaguucucua ccgacaccaa 180

uccaggugcg ucugccacca aguuggcuac ccuuccuagg ggcgcuuucg cgcuugcuca 240

ccauuagauu accuggaaac cagccauuca gguuggaguu uccccaggcu cuuuugugug 300

ggcauuagcg gcuugugguu uuugcacaaa aucuaagcua cuuaccguuc cucugaccau 360

ccaccacuuc uauagacagc acugauuacc guaggguuua agucacaccg gucugcaccg 420

cccgucagcg gacacauuac ccagcauagc acuccuugca ccgcgccuag guaggauaaa 480

acccccuacc gggugacucu uaaggcguuu ccuccacggg auagccacua gucacuaggu 540

guaagugauc ugaucugggc guauuguguu gcgcaagugu gauacccaua ggagcgugga 600

auccuauucu gcggcucagu gccugauaua gcugugaaau ggccaagaac aaguccaagc 660

gcgacgcuau ugcguugccu gaaaauguac caccaccucu gcaacuuuuc auucauguug 720

cagcugcuga agagggucac ccuaagguua cuacuuaccu uggcaacuau aaccucuaug 780

ccaccaaggc uccgccuggc gugcagguuc uuagugcuaa aaccucucuu acugacuuug 840

agaaugucuu uggagcucaa cccaccuugc gaucaauucg uaaucugguu ugugaggcuc 900

gcucggcuga auggacaacu uccaagaaug cuuuugcacu caaagccacu caacuugacu 960

acucugaugc cguuuugagg gcaaugauuc guuucugccc uccaaaggug uccacacucg 1020

cugccuuugc ucuuuuuggc cgauugguua aaauugagga caaggaacuu gcugaguuag 1080

cucgugauac ugcccuugag uuggcguaca cggcuaaaau ugguacaucu cuugcugaca 1140

cgagaucugu cucacuuauu cauaaggacg cuuaucuaac ucucaguaau gagguuguug 1200

gcguaacuuu uacugccgca cuuauggcaa aggcuaccac uguuaaugga gcaaugcaau 1260

acucaaacuu uuaccucuac ccucgugcca cuauuaaggu aaccgauggu aaggcugaag 1320

caauugcaac uaagccucug ucugcugcca cuaaaggcaa gcaaaucaca gaggauguca 1380

accuucuccc ugacuaucag cagcugcuug uugaucaagu gacuggcacu gagguuaagg 1440

uuggagcucu aaccuauguu aagaccacug auucgccacc ccuuuacuuu cccaaaguca 1500

aggguggugu uauugguauu gcacuuaagc agcagggcac uguggcuaag aagcucaaug 1560

uagucuucca ugcucaaccu gaugauguuc ugcuagccuu cauacaacuu cagcaauucu 1620

ugaaccguac uucggauuca aguguugaaa uuacugauug ccagaguuau gaaguaucuc 1680

caacugugac ggucaaaauu ggcccgucua aaccugggga ugucaucgug gcuacugaug 1740

aggaauaccu uaaaugcuuu gaaaccccug agguagguag gcucuauaag guuuuccaaa 1800

cucaaucuug ggcuaucauu gagcgugccu ucuccaguuu gaagauccgc guguccaaag 1860

cuuuaucagc auuuauaagu uuucugcaaa accuugcaga uaacuuuacu gcaauaagug 1920

guguugucac ugcacucauu cgugaacucc aggaucuuac ccuggaugug gcgacacgua 1980

ucacuaacau acaauuuguu uaccgugccg guaagcuuau ugucgacacg acaaguguca 2040

uagcuaaacu uuuccagcca uuuugugauu uuauaucacc uuuccuucgg aaaguugcug 2100

guuuugcaau uuacacuguu gguaaucgca ugcuuauguu uaccagcacu ggcaccuuuc 2160

uucucacaaa ggcaacuacu aagauacuca auaaggcaaa guacaucuuu gauguggagc 2220

cugaguaccc aguagaugua acaacaucca aaguuguagu acaugaagca cuccagcaaa 2280

ccgacacuaa gccuacuaga gcucuggagg cuguugaugu cguuguuggu aauacuguac 2340

ugcaaauggc uacugauggc acugcguucu acccaucgga ugguacgcac gccucucuuc 2400

caggauucaa agcagguucg gaugagcuuu ucauaagcuu caacugcgac cucuuugaug 2460

augagacuaa ugcucaaauc aacgaaacac ucgcugcaua ugagcuuaac caacuagugg 2520

cuccagguga uucuacaccg cgucaaauug cgacguuggu ugucgauaca cuugcagaug 2580

cuauaacaga ccacuuuccg gagaaaacca uugaucuacc ugaagacuau caagucuuuu 2640

cugaccauga ugaccuccca cucgcacaau accacauccc ugaucaccug agccuguaua 2700

uucaggcuau ggaaggugaa gaugauagug gugaugaaau auguauugag gacgaugauu 2760

acgacugucc ucaagccgac gaagacacag aaggaguaau uccccaacag ugggaacuuc 2820

cugauguuga uaaauuuuua cucaagaucc aggaacggaa gaccagcagc gacgaaguac 2880

uuagcgucga cgucuauccu aaaccagagc cggucggcaa uguugggauu gacgacagcg 2940

cgucggaaaa gaagccaaau ggggacucag uaccggaucc ugagguccau ccaacacuag 3000

agagugugga uguugaacga ccaaccgaaa cagcaaacca ggcuguugaa gacaaaccuu 3060

cugauaccac cuuugugguu gaugaggaac aauuacaaga aucaacacca gaacaugaac 3120

uccgcuccua ugaaggggag uuugauucug augaugaaau uauuauuccu auaguaccag 3180

uaacaccugc ggauuuaaaa ccacagacua uuacuauaaa ggaguacuuu aagucugaaa 3240

aacuugagac uauuaacgaa ggauccacag agucaguuac acaaucugac gauucguuug 3300

acgagucauu uguugaugcu gagucugaug auccacaaga uccugcugua uaugaugaua 3360

caacaauuau aacggacagc acugauguag gcgaugagcc ugagacaacu cuagcuacca 3420

ucguuaacac accucugaca cucgauaaua acuugccacc ugaagccauu aaacaaccca 3480

gcccaacuaa gguugaguua guuguuggug aauuggcgag uauuaaauuu gacaauucug 3540

uucuagucaa cccugcuaau gcgcaauuaa caaauggcgg uggagcugcu cgugcaauug 3600

caaaauuagc ugguccaaaa uaucaagagu acuguaauag uguggcuccu aucucaggac 3660

cgcuuaccac ggacucuuuu gaugccaaga aauuuggugu agccugcauc uugcauguag 3720

ugccacccaa agguucugac ccuaauguac aagaacuccu guaucaagcu uacaagagua 3780

uccuuacuga accagcacac uauguuauac cuauacuagg ugcugguauc uuuggaugca 3840

acccagucca cucucuggau gcguucagga aagcaugucc aagugacaua ggucguguca 3900

cccuugucac uaugaacaaa aaccauuugc agguguggga ugcucucaau aggaccauug 3960

uacgcaccac uacugacuau gaucaaguua ccaccaaggc ccuuacaccc cagggagugu 4020

uagaagccaa ucucuuugau ggugaggacu uuguucaaga accaaaaccc ggucaaaucu 4080

accuugaggu uacugaagaa guucagaacc aagccaagga acuugaccuu aaccuucagc 4140

aauacugcgu cuaccugaag acuugccacc auaaaugggu ugugagucgu acgaacgggu 4200

ugaugcaucu aaaacaaaaa gauaacaauu guuuuguuag ugcaggugua aaccuguuuc 4260

aaaacacugc uuaucaacuu agaccugcua uugaugcucu cuauagggag uaucuuaaug 4320

guaauccaaa uagauuuguu gcuuggaucu acgcauccac uaaccgucgu guuggugaga 4380

uggguugucc acagcaaguu auuucuuugc ucguuaguaa cucugacgca gcauuuucag 4440

caacuacagc cuguuguaac accuacuuua accacacagg uguuauuuca guagcucgug 4500

aauaugaccc aauacaacca aaggucuacu gcaugaagug ugaugugugg acucccuuua 4560

caccccagag uggaaaaggu gcaguugcaa uugguauuuc ugcagaugaa ccuaccgguc 4620

cugccauuaa auuugccgca gcucacugcu gguacacuaa uggcaagaaa acaguuaaug 4680

gcuaugacac uaaagcuaau guuguagcua ccuaucauag guuugacgug ccuaagccuc 4740

aacuugucga ggacgugguu gcgcugccua cuaaaaauga cuuugaaguu cucaauguug 4800

aagaacugcc gcaggauagu gugcuccauu uggacccacc uccuguacag gccuuacaac 4860

cuaaggcuaa ccaacacauu gagauucuag aaaacccaga uuaucuggac auuuuggauc 4920

uuuggauucg uaaacccaaa uucauccucg uaaagucgug gaguguuuug gguagagcac 4980

uauguaaggc agguaaaguu gucuuuguca gugcuucgcu uuugacccgu uucuacaauu 5040

accuuguaga gauuggugcu cuugacucaa caauaagguu gucagucgau cuuaccugua 5100

aauuuguuag aacgguucuc ccaucgucua acacuguaca caaaacuugu cuuggucugu 5160

auuauucagc ccagacacuu uuuguuucuu uagcaccauu ccuuauguua ccagcuguag 5220

uuagucugcu uaauucaggc uauacaauug gcacauauuu guaugcaaaa acuggcuggc 5280

cuuguaauua caaugccacg caacacuuug auuauaauuc uuacugugca ggugacuugg 5340

uuugucaagc cuguuuugac ggucaagacu cccuacauuu guauccgcau uuacguguua 5400

aucagcagcc ccuucagacc acugacuaca cuguuuaugc gcuuucacua auacuacuau 5460

uagcuaacau gacucuuguc augggcacgc uaauaguuac uuucuuugug aacuucuaug 5520

gugugcaaau accauuuuau gguacacuuu ugauagauua ucaauccgca cuggugauua 5580

cuuucucagu guacuacuuu uauaagguaa ugaaguuuuu ccgccaucuc acacauggau 5640

guaaaauucc aacgugugug guaugugcca aacuucguac cccaccuacu auaacaguug 5700

agacugucgu ucagggcagg aaauacccau cuguuauuga aacaaauggc ggguuuacaa 5760

uuuguaaaga acacaacuuc uauugcaagg acugcucuuu acaaacaccc ggcacuuuca 5820

ucccgacaga agcuauugag ucgcucucac gagcuaccag gcuuaguguc aaaccaacag 5880

caccagcauu cuuacuugcu agagauguug agugccaaac ugauguuguc guugcucgcg 5940

caaugcauaa ccaaaaugcg caugugugca uuucaaaaua cucugauauc cguaccguug 6000

accaacuacu uaagccuacu ccacuguuuu cauacacucc cgauguuauc aucgcggcag 6060

acuuugacaa cagagguagu cuuaagacag cuaaagaauu agcugugguu uugucaaugg 6120

accuuaaacg uacuauaauu aucauugauc aggccuauuc uagaccuauu gauaauuauc 6180

aggaaguugc uucucguauu gagaaguauu acccaguugc aaagaucaca cccacaggug 6240

acaucuuuac agacauuaag caagcgacca auggccaagc uagugacucu gcuauuaaug 6300

cagcuguucu ggcuguccag cgcggucuug auuuuacaau ugacaacccu aacaacauau 6360

uaccacauua cgccuuugac uuuucaaccc ucaaugcaga agaccagucu accauuuugg 6420

agagugguug ugcuaaaggc aaucucaagg gcacuaaugu ugguguuguu cuuucagcua 6480

gccuuguuac acgucuuagu cagcaggcua uacgugugau ugcuaaugcu gcuucacgua 6540

augguguuac augcgcuguu acuccuucua cacuuguuau gcgugggaau auugcaacac 6600

agcccuugac ucgcaucaaa gcuggugcac cucccaugcg ucaaaaaauu uuauguguua 6660

uccuggcacu ugcuauugug uacuuugcug cuauggcuuu uggcuuuuug gcaagucaaa 6720

uuacgcuuaa uacagugccu acgauuaaau cugauauccg cgccucuacc uucuacguug 6780

uuagagaugg agucuuggau acuguucguu caaaugacaa gugcuuugca aauaaguuuu 6840

uggcauuuga uagcuucauu caagcaccuu acacuaauuc accugacugu ccaguuguug 6900

ugggaguugu ugauguaacg acgcacucua uuccuggaau uccagcaggu gucauucaua 6960

gagacggucu cauacuuaac auuuaugaac agucucuuua ugaaacucau cagcgucagu 7020

cuaugguuag ggaugcguug ucacucaaga cagcaaaucu cuuuaaccua ggcaagcgug 7080

uuguaguagg auacacucaa caugaaguug uuguggguac cuccuauuuu aauucuccug 7140

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uuuaaaaaua uaaccacugg caaaacccuu caggucuuac cgugugaaac cccuucucaa 20880

cugauuguga uaaacaacac cguugucggu gcuaucacau ccaguaauuc aacugaaaau 20940

aauagguuua cuacuacuau ugucacaccu acuuucuuuu auuccacaaa ugccaccacu 21000

uucaacugca cuaagccugu uuuguccuau ggaccuauca gcguguguag ugauggugca 21060

auugugggaa cauccacauu acagaauacu cgaccaucca uaguuucacu auacgauggc 21120

gaaguugaaa uaccaucugc auuuucucuu uccguucaga cggaguacuu gcaaguucaa 21180

gcagagcaag uuauaguuga uuguccucag uauguaugca auggcaacag ccguugucua 21240

caauuacugg cacaauacac cucagcuugc ucuaacauug aagcagcucu gcauuccucu 21300

gcacaguugg auagcagaga gauuauaaau auguuucaaa caucaacaca guccuugcag 21360

uuagcuaaua uuaccaacuu caagggugac uacaauuuua gcagcauacu aaccaccaga 21420

auugguggca gaucugcuau ugaagaccuu cuuuuuaaua aaguuguuac uaguggccuu 21480

ggcacuguug aucaggacua caaauccugc ucuagagaca uggccaucgc ugacuuaguu 21540

uguucccagu auuacaaugg caucaugguu cuaccuggug uuguugaugc ugagaaaaug 21600

gcaauguaua cuggcucucu uacuggagcu augguauuug gaggacugac ugccgcagcg 21660

gcaauaccau uugccacggc aguacaagcu cgccucaauu augucgcacu gcaaacaaau 21720

guacuacaag aaaaccagaa aauucuugca gaaucauuua accaagcagu uggcaauaua 21780

ucacuugcac uaucuucugu uaaugaugcc auccagcaaa cuucugaggc ucuuaacacc 21840

guagcuauug cuauuaaaaa gauucaaaca guuguuaacc agcagggcga ggcauuauca 21900

caccugacug cacagcuguc aaacaauuuu caagcaauuu cgacuucuau ucaagacauu 21960

uacaaccguc uugaggaagu agaggcuaac cagcaaguug accgucucau cacaggacgg 22020

uuggcugcac uuaaugcaua uguuacucag uuacucaauc agaugucuca gauuagacaa 22080

ucucgauugu uagcucagca aaagauuaau gaguguguca aaucacaguc guccagauac 22140

gguuucugug gaaauggcac acacaucuuc ucacuuacac agacugcacc aaauggcaua 22200

uuuuucaugc augcagugcu aguacccaac aaauucacac gugucaacgc uucugccggc 22260

auuugugugg auaauacgag aggcuacuca uugcagccuc aacuuauacu cuaccaguuu 22320

aauaacuccu ggagaguuac accuagaaau auguaugaac ccagacugcc ccggcaggcu 22380

gauuucauac aauuaacuga uugcagcguu acuuuuuaca acaccaccgc ugcuaaucuu 22440

cccaauauua ucccugacau uauagauguc aaucaaacag ucagugauau uauugacaau 22500

uuaccuacag caacaccucc ucagugggau guugguaucu auaacaacac uauucucaac 22560

cucaccguug agauuaauga ucuacaagag cggucuaaaa accucucaca gauugcagau 22620

cguuuacaaa auuauauuga caaucuuaac aauacucuag uugaccuuga auggcucaac 22680

agaguggaaa cuuaccuuaa auggccgugg uauauauggc uugccauugc ccuggcucuu 22740

auugcauuug ugacaauccu cauaacaauc uuucuuugua cugguuguug uggugguugc 22800

uuugguuguu guggcgguug uuuuggccuu uucucuaaga agaaaaggua uaccgacgac 22860

caaccaacac cguccuuuaa guuuaaggaa ugguagucga cgacugggcc guuaccaucc 22920

cuggacaaua uauuauugcu auacuaguug ucaucugcau ugguguggca cuacuuuuua 22980

uuaauacuug cuuagcuugu guuaaauuau uuuacaagug cuaccuaggg gcagcauauc 23040

uuguuaggcc uauuauagug uacuacucca agccgaaccc cguaccugag gaugaguuug 23100

uaaaaguaca ccaauuuccu agaaacacuc acuaugucug acgcagaaga guggcaaauu 23160

auuguuuuca uugcgaucau augggcgcuu ggcgucaucc uccagggagg cuaugccacg 23220

cguaaucgug ugaucuaugu uauuaaacuu auucugcuuu ggcugcucca acccuucacc 23280

cuagugguga ccauuuggac cgcagucgac agaucaucua agaaggacgc aguuuucauu 23340

guguccauaa uuuuugccgu acugaccuuc auauccuggg ccaaguacug guaugacuca 23400

auucguuuau uaaugaaaac cagaucugca ugggcacucu caccugagag uagacuccuu 23460

gcagggauua uggauccaau ggguacaugg aggugcauuc ccaucgacca cauggcucca 23520

auucucacac cagucguuaa gcauggcaag cucaagcuac augggcaaga gcuggccaau 23580

ggcauaucag ucagaaaucc gccacaggau auggugauag ugucaccaag ugacaccuuu 23640

cacuacacuu uuaagaaacc uguggaauca aacaacgauc cagaauuugc uguucugaua 23700

uaccagggug accgcgcuuc aaacgcugga cuucacacca uaaccacuuc aaaggccggu 23760

gacgcucgcc uguauaagua uauguaaugu gcaacugcca ucugcagcug cgagauuuau 23820

auagauugug caauaagcgg cacaucagaa gagaggaugu uccugagcuu auugacccuc 23880

ucguuaaaac ucgcuguuuu gcuuacaguc ucgugguucu ugcuaaugcu aauccaauug 23940

cauuuagcau acuaccucgg aaaauucuua ucaaugguga gccuuuacug cuugaauaug 24000

guagcauaua ugguaaagac uuuaucauuc gaccaucgcu ccaagucauu cuugaagaug 24060

aauuaaauua aaguuuugac accaaucuau cauggcugca ccaguagucc cuacuacuga 24120

cgcgucuugg uuucaggugc ucaaagcuca aaacaaaaag gcuacucauc cucaguuucg 24180

uggcaaugga guuccgcuua acuccgccau caaacccguu gaaaaccaug gcuacuggcu 24240

gcguuacacc agacaaaagc caggugguac ucccauuccu ccauccuaug ccuuuuauua 24300

uacuggcaca ggucccagag gaaaucuuaa guauggugaa cucccuccua augauacccc 24360

agcaaccacu cguguuacuu ggguuaaggg uucgggagcu gacacuucua uuaaaccuca 24420

uguugccaaa cgcaacccca acaauccuaa acaucagcug cuaccucucc gauucccaac 24480

cggagauggc ccagcucaag guuucagagu ugaccccuuc aacgcuagag gaagaccuca 24540

ggagcgugga aguggcccaa gaucucaauc uguuaacucc agaggcacag gcaaucagcc 24600

caggaaacgc gaccaaucug caccagcugc gguacgucgu aagacccagc aucaagcucc 24660

caagcggacu uuacccaagg guaaaaccau uucucaggua uuuggcaacc ggucucguac 24720

uggugccaau gucggcucug cagacacuga gaagacgggu auggcugauc cucgcaucau 24780

ggcucuagcc agacaugugc cugguguuca ggaaaugcuu uuugcuggcc accuugagag 24840

caacuuucag gcgggggcaa uaacccuuac cuucuccuac ucaaucacag ucaaggaggg 24900

uucuccugac uaugagagac uuaaggaugc gcucaauacg gucguuaacc agaccuauga 24960

gccaccuacc aaaccaacua aggacaagaa gccugacaaa caagaccagu cugcuaaacc 25020

caaacagcag aagaaaccua aaaagguaac ucugccagca gacaaacagg auugggagug 25080

ggaugaugcu uuugagauaa agcaggaauc agcagcguag acaucaaucu augucuguua 25140

aacccaccca acuccacuca aauaucucuu ugguuccaga gagucguagu guauagccag 25200

agagccaguc agagggcgcu aucaugcaaa cuagggcugg cuacucuagc acagaaucac 25260

aucccgauaa ucaacagugc uagaagguug auuauaccau uuaauaugcc gaggccacgc 25320

ggaguacgau cgaggguaca gcauaaucuc aacuuuuguu gagccacaau uuuaauccua 25380

auuggagaag gccaaaggac uguacuacuu uugugggugu agcagucgcc cagugggaaa 25440

gcgccaacua gguuacaauu guggugggga caaauuaggg gaaauuaaau uggcuuau 25498

<210> 11

<211> 3471

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine Deltacorona Virus BIV-Isolate 5.0327 Spike Antigen cDNA

<400> 11

atgcagagag ctctattgat tatgacctta ttttgtctcg ttcgagcaaa gtttgctgat 60

gatctactcg atttgctcac cttcccgggt gcacatcgct tcttacataa acccacgagg 120

aattccagca gtctctactc gcgggctaat aataattttg atgttggcgt tcttcctggc 180

taccccacta agaacgttaa cctcttctca ccacttacta actccacttt gcccattaat 240

ggccttcatc ggagttacca accactcatg ctgaattgtc ttactaaaat aactaaccac 300

actctcagca tgtatctcct acctagtgag atacaaactt atagctgcgg cggtgccatg 360

gttaaatacc agacacatga tgcagttcgt atcattttag acctcactgc cactgaccac 420

atctctgttg aagtcgttgg ccaacatggt gaaaattatg tgtttgtttg cagtgagcag 480

tttaactaca ccactgcatt acacaactct accttcttct cacttaattc tgagctttat 540

tgctttacta ataacaccta cttaggtatt cttccacctg atttaactga ctttacggtc 600

taccgtactg gtcagttcta tgctaatggt taccttttag gtactttacc tattacggtt 660

aactatgtta ggttgtatcg gggtcatttt agtgcccact ttgcccttgc aaacctaacc 720

gatacactca taacacttac caatactact atatcgcaaa tcacttattg tgataagtct 780

gtagttgatt caatagcatg ccagcgctct tctcacgaag tggaggatgg gttttactcc 840

gaccctaaat ctgccgttag agctaggcaa cgtactattg ttacactacc taagctccct 900

gagcttgaag tagtgcagtt aaatatttct gcacacatgg attttggcga agccagactt 960

gacagcgtta ccatcaatgg taacacatcc tattgtgtca ctaagcctta cttcaggctt 1020

gaaactaact ttatgtgtac aggttgcact atgaatctgc gcactgatac ctgtagtttt 1080

gacctgtcag cagtaaacaa tggcatgtca ttctctcaat tctgtctaag cactgaatct 1140

ggtgcttgtg agatgaaaat tattgttacc tacgtatgga attacttgct aaggcagcgt 1200

ttgtatgtta ctgctgtaga gggccagact cacactggaa ccacttcagt acatgcaaca 1260

gacacttcta gtgtaatcac tgatgtctgc actgactaca ctatctatgg agtctctggt 1320

actggcatta ttaagccatc agatctctta ttgcacaatg gcatagcatt cacctctcca 1380

acaggtgagc tttatgcatt taaaaatata accactggca aaacccttca ggtcttaccg 1440

tgtgaaaccc cttctcaact gattgtgata aacaacaccg ttgtcggtgc tatcacatcc 1500

agtaattcaa ctgaaaataa taggtttact actactattg tcacacctac tttcttttat 1560

tccacaaatg ccaccacttt caactgcact aagcctgttt tgtcctatgg acctatcagc 1620

gtgtgtagtg atggtgcaat tgtgggaaca tccacattac agaatactcg accatccata 1680

gtttcactat acgatggcga agttgaaata ccatctgcat tttctctttc cgttcagacg 1740

gagtacttgc aagttcaagc agagcaagtt atagttgatt gtcctcagta tgtatgcaat 1800

ggcaacagcc gttgtctaca attactggca caatacacct cagcttgctc taacattgaa 1860

gcagctctgc attcctctgc acagttggat agcagagaga ttataaatat gtttcaaaca 1920

tcaacacagt ccttgcagtt agctaatatt accaacttca agggtgacta caattttagc 1980

agcatactaa ccaccagaat tggtggcaga tctgctattg aagaccttct ttttaataaa 2040

gttgttacta gtggccttgg cactgttgat caggactaca aatcctgctc tagagacatg 2100

gccatcgctg acttagtttg ttcccagtat tacaatggca tcatggttct acctggtgtt 2160

gttgatgctg agaaaatggc aatgtatact ggctctctta ctggagctat ggtatttgga 2220

ggactgactg ccgcagcggc aataccattt gccacggcag tacaagctcg cctcaattat 2280

gtcgcactgc aaacaaatgt actacaagaa aaccagaaaa ttcttgcaga atcatttaac 2340

caagcagttg gcaatatatc acttgcacta tcttctgtta atgatgccat ccagcaaact 2400

tctgaggctc ttaacaccgt agctattgct attaaaaaga ttcaaacagt tgttaaccag 2460

cagggcgagg cattatcaca cctgactgca cagctgtcaa acaattttca agcaatttcg 2520

acttctattc aagacattta caaccgtctt gaggaagtag aggctaacca gcaagttgac 2580

cgtctcatca caggacggtt ggctgcactt aatgcatatg ttactcagtt actcaatcag 2640

atgtctcaga ttagacaatc tcgattgtta gctcagcaaa agattaatga gtgtgtcaaa 2700

tcacagtcgt ccagatacgg tttctgtgga aatggcacac acatcttctc acttacacag 2760

actgcaccaa atggcatatt tttcatgcat gcagtgctag tacccaacaa attcacacgt 2820

gtcaacgctt ctgccggcat ttgtgtggat aatacgagag gctactcatt gcagcctcaa 2880

cttatactct accagtttaa taactcctgg agagttacac ctagaaatat gtatgaaccc 2940

agactgcccc ggcaggctga tttcatacaa ttaactgatt gcagcgttac tttttacaac 3000

accaccgctg ctaatcttcc caatattatc cctgacatta tagatgtcaa tcaaacagtc 3060

agtgatatta ttgacaattt acctacagca acacctcctc agtgggatgt tggtatctat 3120

aacaacacta ttctcaacct caccgttgag attaatgatc tacaagagcg gtctaaaaac 3180

ctctcacaga ttgcagatcg tttacaaaat tatattgaca atcttaacaa tactctagtt 3240

gaccttgaat ggctcaacag agtggaaact taccttaaat ggccgtggta tatatggctt 3300

gccattgccc tggctcttat tgcatttgtg acaatcctca taacaatctt tctttgtact 3360

ggttgttgtg gtggttgctt tggttgttgt ggcggttgtt ttggcctttt ctctaagaag 3420

aaaaggtata ccgacgacca accaacaccg tcctttaagt ttaaggaatg g 3471

<210> 12

<211> 1157

<212> PRT

<213> Porcine Deltacorona Virus

<400> 12

Met Gln Arg Ala Leu Leu Ile Met Thr Leu Phe Cys Leu Val Arg Ala

1 5 10 15

Lys Phe Ala Asp Asp Leu Leu Asp Leu Leu Thr Phe Pro Gly Ala His

20 25 30

Arg Phe Leu His Lys Pro Thr Arg Asn Ser Ser Ser Leu Tyr Ser Arg

35 40 45

Ala Asn Asn Asn Phe Asp Val Gly Val Leu Pro Gly Tyr Pro Thr Lys

50 55 60

Asn Val Asn Leu Phe Ser Pro Leu Thr Asn Ser Thr Leu Pro Ile Asn

65 70 75 80

Gly Leu His Arg Ser Tyr Gln Pro Leu Met Leu Asn Cys Leu Thr Lys

85 90 95

Ile Thr Asn His Thr Leu Ser Met Tyr Leu Leu Pro Ser Glu Ile Gln

100 105 110

Thr Tyr Ser Cys Gly Gly Ala Met Val Lys Tyr Gln Thr His Asp Ala

115 120 125

Val Arg Ile Ile Leu Asp Leu Thr Ala Thr Asp His Ile Ser Val Glu

130 135 140

Val Val Gly Gln His Gly Glu Asn Tyr Val Phe Val Cys Ser Glu Gln

145 150 155 160

Phe Asn Tyr Thr Thr Ala Leu His Asn Ser Thr Phe Phe Ser Leu Asn

165 170 175

Ser Glu Leu Tyr Cys Phe Thr Asn Asn Thr Tyr Leu Gly Ile Leu Pro

180 185 190

Pro Asp Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr Ala

195 200 205

Asn Gly Tyr Leu Leu Gly Thr Leu Pro Ile Thr Val Asn Tyr Val Arg

210 215 220

Leu Tyr Arg Gly His Phe Ser Ala His Phe Ala Leu Ala Asn Leu Thr

225 230 235 240

Asp Thr Leu Ile Thr Leu Thr Asn Thr Thr Ile Ser Gln Ile Thr Tyr

245 250 255

Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln Arg Ser Ser His

260 265 270

Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser Ala Val Arg Ala

275 280 285

Arg Gln Arg Thr Ile Val Thr Leu Pro Lys Leu Pro Glu Leu Glu Val

290 295 300

Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly Glu Ala Arg Leu

305 310 315 320

Asp Ser Val Thr Ile Asn Gly Asn Thr Ser Tyr Cys Val Thr Lys Pro

325 330 335

Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly Cys Thr Met Asn

340 345 350

Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala Val Asn Asn Gly

355 360 365

Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ser Gly Ala Cys Glu

370 375 380

Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu Leu Arg Gln Arg

385 390 395 400

Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr Gly Thr Thr Ser

405 410 415

Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp Val Cys Thr Asp

420 425 430

Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile Lys Pro Ser Asp

435 440 445

Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro Thr Gly Glu Leu

450 455 460

Tyr Ala Phe Lys Asn Ile Thr Thr Gly Lys Thr Leu Gln Val Leu Pro

465 470 475 480

Cys Glu Thr Pro Ser Gln Leu Ile Val Ile Asn Asn Thr Val Val Gly

485 490 495

Ala Ile Thr Ser Ser Asn Ser Thr Glu Asn Asn Arg Phe Thr Thr Thr

500 505 510

Ile Val Thr Pro Thr Phe Phe Tyr Ser Thr Asn Ala Thr Thr Phe Asn

515 520 525

Cys Thr Lys Pro Val Leu Ser Tyr Gly Pro Ile Ser Val Cys Ser Asp

530 535 540

Gly Ala Ile Val Gly Thr Ser Thr Leu Gln Asn Thr Arg Pro Ser Ile

545 550 555 560

Val Ser Leu Tyr Asp Gly Glu Val Glu Ile Pro Ser Ala Phe Ser Leu

565 570 575

Ser Val Gln Thr Glu Tyr Leu Gln Val Gln Ala Glu Gln Val Ile Val

580 585 590

Asp Cys Pro Gln Tyr Val Cys Asn Gly Asn Ser Arg Cys Leu Gln Leu

595 600 605

Leu Ala Gln Tyr Thr Ser Ala Cys Ser Asn Ile Glu Ala Ala Leu His

610 615 620

Ser Ser Ala Gln Leu Asp Ser Arg Glu Ile Ile Asn Met Phe Gln Thr

625 630 635 640

Ser Thr Gln Ser Leu Gln Leu Ala Asn Ile Thr Asn Phe Lys Gly Asp

645 650 655

Tyr Asn Phe Ser Ser Ile Leu Thr Thr Arg Ile Gly Gly Arg Ser Ala

660 665 670

Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Ser Gly Leu Gly Thr

675 680 685

Val Asp Gln Asp Tyr Lys Ser Cys Ser Arg Asp Met Ala Ile Ala Asp

690 695 700

Leu Val Cys Ser Gln Tyr Tyr Asn Gly Ile Met Val Leu Pro Gly Val

705 710 715 720

Val Asp Ala Glu Lys Met Ala Met Tyr Thr Gly Ser Leu Thr Gly Ala

725 730 735

Met Val Phe Gly Gly Leu Thr Ala Ala Ala Ala Ile Pro Phe Ala Thr

740 745 750

Ala Val Gln Ala Arg Leu Asn Tyr Val Ala Leu Gln Thr Asn Val Leu

755 760 765

Gln Glu Asn Gln Lys Ile Leu Ala Glu Ser Phe Asn Gln Ala Val Gly

770 775 780

Asn Ile Ser Leu Ala Leu Ser Ser Val Asn Asp Ala Ile Gln Gln Thr

785 790 795 800

Ser Glu Ala Leu Asn Thr Val Ala Ile Ala Ile Lys Lys Ile Gln Thr

805 810 815

Val Val Asn Gln Gln Gly Glu Ala Leu Ser His Leu Thr Ala Gln Leu

820 825 830

Ser Asn Asn Phe Gln Ala Ile Ser Thr Ser Ile Gln Asp Ile Tyr Asn

835 840 845

Arg Leu Glu Glu Val Glu Ala Asn Gln Gln Val Asp Arg Leu Ile Thr

850 855 860

Gly Arg Leu Ala Ala Leu Asn Ala Tyr Val Thr Gln Leu Leu Asn Gln

865 870 875 880

Met Ser Gln Ile Arg Gln Ser Arg Leu Leu Ala Gln Gln Lys Ile Asn

885 890 895

Glu Cys Val Lys Ser Gln Ser Ser Arg Tyr Gly Phe Cys Gly Asn Gly

900 905 910

Thr His Ile Phe Ser Leu Thr Gln Thr Ala Pro Asn Gly Ile Phe Phe

915 920 925

Met His Ala Val Leu Val Pro Asn Lys Phe Thr Arg Val Asn Ala Ser

930 935 940

Ala Gly Ile Cys Val Asp Asn Thr Arg Gly Tyr Ser Leu Gln Pro Gln

945 950 955 960

Leu Ile Leu Tyr Gln Phe Asn Asn Ser Trp Arg Val Thr Pro Arg Asn

965 970 975

Met Tyr Glu Pro Arg Leu Pro Arg Gln Ala Asp Phe Ile Gln Leu Thr

980 985 990

Asp Cys Ser Val Thr Phe Tyr Asn Thr Thr Ala Ala Asn Leu Pro Asn

995 1000 1005

Ile Ile Pro Asp Ile Ile Asp Val Asn Gln Thr Val Ser Asp Ile

1010 1015 1020

Ile Asp Asn Leu Pro Thr Ala Thr Pro Pro Gln Trp Asp Val Gly

1025 1030 1035

Ile Tyr Asn Asn Thr Ile Leu Asn Leu Thr Val Glu Ile Asn Asp

1040 1045 1050

Leu Gln Glu Arg Ser Lys Asn Leu Ser Gln Ile Ala Asp Arg Leu

1055 1060 1065

Gln Asn Tyr Ile Asp Asn Leu Asn Asn Thr Leu Val Asp Leu Glu

1070 1075 1080

Trp Leu Asn Arg Val Glu Thr Tyr Leu Lys Trp Pro Trp Tyr Ile

1085 1090 1095

Trp Leu Ala Ile Ala Leu Ala Leu Ile Ala Phe Val Thr Ile Leu

1100 1105 1110

Ile Thr Ile Phe Leu Cys Thr Gly Cys Cys Gly Gly Cys Phe Gly

1115 1120 1125

Cys Cys Gly Gly Cys Phe Gly Leu Phe Ser Lys Lys Lys Arg Tyr

1130 1135 1140

Thr Asp Asp Gln Pro Thr Pro Ser Phe Lys Phe Lys Glu Trp

1145 1150 1155

<210> 13

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967167A PDCoV SPIKE FORWARD PRIMER

<400> 13

ggatccatgc agagagctct attgattatg acc 33

<210> 14

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967167B PDCoV SPIKE REVERSE PRIMER

<400> 14

ggtcgagccg cctctgccac caattctggt ggttagtaag ct 42

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967167C FC FORWARD PRIMER

<400> 15

tggtggcaga ggcggctcga ccaagaccaa g 31

<210> 16

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> P2968014E FC FORWARD PRIMER

<400> 16

gcggccgcct acttgcccgg cgtcttcgaa atggat 36

<210> 17

<211> 2718

<212> DNA

<213> Artificial Sequence

<220>

<223> PDCoV S1-IgG2a Fc CDS cDNA Expression Casette

<400> 17

atgcagagag ctctattgat tatgacctta ctttgtctcg ttcgagcaaa gtttgctgat 60

gatctactcg atttgctcac cttcccgggt gcacatcgct tcttacataa acccacgagg 120

aattccagca gtctctactc gcgggctaat aataattttg atgttggcgt tcttcctggc 180

taccccacta agaacgttaa cctcttctca ccacttacta actccacttt gcccattaat 240

ggccttcatc ggagttacca accactcatg ctgaattgtc ttactaaaat aactaaccac 300

actctcagca tgtatctcct acctagtgag atacaaactt atagctgcgg cggtgccatg 360

gttaaatacc agacacatga tgcagttcgt atcattttag acctcactgc cactgaccac 420

atctctgttg aagtcgttgg ccaacatggt gaaaattatg tgtttgtttg cagtgagcag 480

tttaactaca ccactgcatt acacaactct accttcttct cacttaattc tgagctttat 540

tgctttacta ataacaccta cttaggtatt cttccacctg atttaactga ctttacggtc 600

taccgtactg gtcagttcta tgctaatggt taccttttag gtactttacc tattacggtt 660

aactatgtta ggttgtatcg gggtcatttg tctgccaata gtgcctactt tgcccttgca 720

aacctaaccg atacactcat aacacttacc aatactacta tatcgcaaat cacttattgt 780

gataagtcag tagttgattc aatagcatgc cagcgctctt ctcacgaagt ggaggatggg 840

ttttactccg accctaaatc tgccgttaga gctaggcaac gtactattgt tacactacct 900

aagctccctg agcttgaagt agtgcagtta aatatttctg cacacatgga ttttggcgaa 960

gccagacttg acagcgttac catcaatggt aacacatcct attgtgtcac taagccttac 1020

ttcaggcttg aaactaactt tatgtgtaca ggttgcacta tgaatctgcg cactgatacc 1080

tgtagttttg acctgtcagc agtaaacaat ggcatgtcat tctctcaatt ctgtctaagc 1140

actgaatctg gtgcttgtga gatgaaaatt attgttacct acgtatggaa ttacttgcta 1200

aggcagcgtt tgtatgttac tgctgtagag ggccagactc acactggaac cacttcagta 1260

catgcaacag acacttctag tgtaatcact gatgtctgca ctgactacac tatctatgga 1320

gtctcaggta ctggcattat taagccatca gatctcttat tgcacaatgg catagcattc 1380

acctctccaa caggtgagct ttatgcattt aaaaatataa ccactggcaa aacccttcag 1440

gtcttaccgt gtgaaacccc ttctcaactg attgtgataa acaacaccgt tgtcggtgct 1500

atcacatcca gtaattcaac tgaaaataat aggtttacta ctactattgt cacacctact 1560

ttcttttatt ccacaaatgc caccactttc aactgcacta agcctgtttt gtcctatgga 1620

cctatcagcg tgtgtagtga tggtgcaatt gtgggaacat ccacattaca gaatactcga 1680

ccatccatag tttcactata cgatggcgaa gttgaaatac catctgcatt ttctctttcc 1740

gttcagacgg agtacttgca agttcaagca gagcaagtta tagttgattg tcctcagtat 1800

gtatgcaatg gcaacagccg ttgtctacaa ttactggcac aatacacctc agcttgctct 1860

aacattgaag cagctctgca ttcctctgca cagttggata gcagagagat tataaatatg 1920

tttcaaacat caacacagtc cttgcagtta gctaatatta ccaacttcaa gggtgactac 1980

aattttagca gcttactaac caccagaatt ggtggcagag gcggctcgac caagaccaag 2040

cccccctgtc ctatttgccc ggcatgcgag agtccgggtc cgtccgtctt tattttcccc 2100

ccaaagccaa aagatacgct gatgatcagc cgcacccccc aggtcacatg cgtggttgta 2160

gatgtaagcc aagaaaatcc cgaagtgcag ttttcctggt atgtcgatgg cgtggaggtt 2220

cacacagcac agacgcggcc gaaggaagag cagtttaatt ccacgtaccg agtggtgagt 2280

gtgctgccta ttcagcatca ggattggttg aacggaaagg agttcaagtg caaggtgaac 2340

aacaaagacc tgcccgcacc aatcactcgc ataatcagca aggctaaggg acagacacgc 2400

gagccacagg tgtacaccct gccaccgcat gcagaagagc tgagccgatc caaggtatcc 2460

ataacctgcc tggtgatcgg attctacccc cccgatatcg acgtcgagtg gcagaggaat 2520

ggccagccgg agccagaggg caactaccgg accacgcctc cgcagcagga tgtcgatggt 2580

acatattttc tgtacagcaa atttagcgtg gacaaggcca gttggcaggg cggcggtatc 2640

ttccagtgtg ctgtcatgca cgaggcactc cacaatcatt acacccagaa atccatttcg 2700

aagacgccgg gcaagtag 2718

<210> 18

<211> 905

<212> PRT

<213> Artificial Sequence

<220>

<223> PDCoV S1-IgG2a Fc Amino Acid Sequence

<400> 18

Met Gln Arg Ala Leu Leu Ile Met Thr Leu Leu Cys Leu Val Arg Ala

1 5 10 15

Lys Phe Ala Asp Asp Leu Leu Asp Leu Leu Thr Phe Pro Gly Ala His

20 25 30

Arg Phe Leu His Lys Pro Thr Arg Asn Ser Ser Ser Leu Tyr Ser Arg

35 40 45

Ala Asn Asn Asn Phe Asp Val Gly Val Leu Pro Gly Tyr Pro Thr Lys

50 55 60

Asn Val Asn Leu Phe Ser Pro Leu Thr Asn Ser Thr Leu Pro Ile Asn

65 70 75 80

Gly Leu His Arg Ser Tyr Gln Pro Leu Met Leu Asn Cys Leu Thr Lys

85 90 95

Ile Thr Asn His Thr Leu Ser Met Tyr Leu Leu Pro Ser Glu Ile Gln

100 105 110

Thr Tyr Ser Cys Gly Gly Ala Met Val Lys Tyr Gln Thr His Asp Ala

115 120 125

Val Arg Ile Ile Leu Asp Leu Thr Ala Thr Asp His Ile Ser Val Glu

130 135 140

Val Val Gly Gln His Gly Glu Asn Tyr Val Phe Val Cys Ser Glu Gln

145 150 155 160

Phe Asn Tyr Thr Thr Ala Leu His Asn Ser Thr Phe Phe Ser Leu Asn

165 170 175

Ser Glu Leu Tyr Cys Phe Thr Asn Asn Thr Tyr Leu Gly Ile Leu Pro

180 185 190

Pro Asp Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr Ala

195 200 205

Asn Gly Tyr Leu Leu Gly Thr Leu Pro Ile Thr Val Asn Tyr Val Arg

210 215 220

Leu Tyr Arg Gly His Leu Ser Ala Asn Ser Ala Tyr Phe Ala Leu Ala

225 230 235 240

Asn Leu Thr Asp Thr Leu Ile Thr Leu Thr Asn Thr Thr Ile Ser Gln

245 250 255

Ile Thr Tyr Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln Arg

260 265 270

Ser Ser His Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser Ala

275 280 285

Val Arg Ala Arg Gln Arg Thr Ile Val Thr Leu Pro Lys Leu Pro Glu

290 295 300

Leu Glu Val Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly Glu

305 310 315 320

Ala Arg Leu Asp Ser Val Thr Ile Asn Gly Asn Thr Ser Tyr Cys Val

325 330 335

Thr Lys Pro Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly Cys

340 345 350

Thr Met Asn Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala Val

355 360 365

Asn Asn Gly Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ser Gly

370 375 380

Ala Cys Glu Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu Leu

385 390 395 400

Arg Gln Arg Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr Gly

405 410 415

Thr Thr Ser Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp Val

420 425 430

Cys Thr Asp Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile Lys

435 440 445

Pro Ser Asp Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro Thr

450 455 460

Gly Glu Leu Tyr Ala Phe Lys Asn Ile Thr Thr Gly Lys Thr Leu Gln

465 470 475 480

Val Leu Pro Cys Glu Thr Pro Ser Gln Leu Ile Val Ile Asn Asn Thr

485 490 495

Val Val Gly Ala Ile Thr Ser Ser Asn Ser Thr Glu Asn Asn Arg Phe

500 505 510

Thr Thr Thr Ile Val Thr Pro Thr Phe Phe Tyr Ser Thr Asn Ala Thr

515 520 525

Thr Phe Asn Cys Thr Lys Pro Val Leu Ser Tyr Gly Pro Ile Ser Val

530 535 540

Cys Ser Asp Gly Ala Ile Val Gly Thr Ser Thr Leu Gln Asn Thr Arg

545 550 555 560

Pro Ser Ile Val Ser Leu Tyr Asp Gly Glu Val Glu Ile Pro Ser Ala

565 570 575

Phe Ser Leu Ser Val Gln Thr Glu Tyr Leu Gln Val Gln Ala Glu Gln

580 585 590

Val Ile Val Asp Cys Pro Gln Tyr Val Cys Asn Gly Asn Ser Arg Cys

595 600 605

Leu Gln Leu Leu Ala Gln Tyr Thr Ser Ala Cys Ser Asn Ile Glu Ala

610 615 620

Ala Leu His Ser Ser Ala Gln Leu Asp Ser Arg Glu Ile Ile Asn Met

625 630 635 640

Phe Gln Thr Ser Thr Gln Ser Leu Gln Leu Ala Asn Ile Thr Asn Phe

645 650 655

Lys Gly Asp Tyr Asn Phe Ser Ser Leu Leu Thr Thr Arg Ile Gly Gly

660 665 670

Arg Gly Gly Ser Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Ala

675 680 685

Cys Glu Ser Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

690 695 700

Asp Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val Val

705 710 715 720

Asp Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val Asp

725 730 735

Gly Val Glu Val His Thr Ala Gln Thr Arg Pro Lys Glu Glu Gln Phe

740 745 750

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp

755 760 765

Trp Leu Asn Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

770 775 780

Pro Ala Pro Ile Thr Arg Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg

785 790 795 800

Glu Pro Gln Val Tyr Thr Leu Pro Pro His Ala Glu Glu Leu Ser Arg

805 810 815

Ser Lys Val Ser Ile Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp

820 825 830

Ile Asp Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Pro Glu Gly Asn

835 840 845

Tyr Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe Leu

850 855 860

Tyr Ser Lys Phe Ser Val Asp Lys Ala Ser Trp Gln Gly Gly Gly Ile

865 870 875 880

Phe Gln Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

885 890 895

Lys Ser Ile Ser Lys Thr Pro Gly Lys

900 905

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194A F Primer PDCoVS BD

<400> 19

ggatccgcca ccatggtatc tg 22

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194B R Primer PDCoVS BD

<400> 20

gcttaaatac atatttagta c 21

<210> 21

<211> 100

<212> DNA

<213> Artificial Sequence

<220>

<223> O2967002 A Primer Gp64 signal sequence

<400> 21

ggatccgcca ccatggtatc tgctattgtt ctttatgttt tgcttgctgc cgccgctcac 60

tcagctttcg cggatgatct actcgatttg ctcaccttcc 100

<210> 22

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> O2967002B AcNPV gp64 C-terminal tail coding sequence

<400> 22

gcggccgctt agtactgacg attgcggttt ctaagaaaga ttgttatgag gattgtcaca 60

aat 63

<210> 23

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967002C F Primer PDCoVS BD

<400> 23

gatgatctac tcgatttgct caccttcc 28

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967002D R Primer PDCoVS BD

<400> 24

cgatggccat gtctctagag caggat 26

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> P2967002E F Primer PDCoVS BD

<400> 25

tgtcctatgg acctatcagc gtgtg 25

<210> 26

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> P2968002F R Primer PDCoVS BD

<400> 26

aagaaagatt gttatgagga ttgtcacaaa t 31

<210> 27

<211> 3390

<212> DNA

<213> Artificial Sequence

<220>

<223> PDCoVS BD CDS Expression Cassette

<400> 27

atggtatctg ctattgttct ttatgttttg cttgctgccg ccgctcactc agctttcgcg 60

gatgatctac tcgatttgct caccttcccg ggtgcacatc gcttcttaca taaacccacg 120

aggaattcca gcagtctcta ctcgcgggct aataataatt ttgatgttgg cgttcttcct 180

ggctacccca ctaagaacgt taacctcttc tcaccactta ctaactccac tttgcccatt 240

aatggccttc atcggagtta ccaaccactc atgctgaatt gtcttactaa aataactaac 300

cacactctca gcatgtatct cctacctagt gagatacaaa cttatagctg cggcggtgcc 360

atggttaaat accagacaca tgatgcagtt cgtatcattt tagacctcac tgccactgac 420

cacatctctg ttgaagtcgt tggccaacat ggtgaaaatt atgtgtttgt ttgcagtgag 480

cagtttaact acaccactgc attacacaac tctaccttct tctcacttaa ttctgagctt 540

tattgcttta ctaataacac ctacttaggt attcttccac ctgatttaac tgactttacg 600

gtctaccgta ctggtcagtt ctatgctaat ggttaccttt taggtacttt acctattacg 660

gttaactatg ttaggttgta tcggggtcat ttgtctgcca atagtgccta ctttgccctt 720

gcaaacctaa ccgatacact cataacactt accaatacta ctatatcgca aatcacttat 780

tgtgataagt cagtagttga ttcaatagca tgccagcgct cttctcacga agtggaggat 840

gggttttact ccgaccctaa atctgccgtt agagctaggc aacgtactat tgttacacta 900

cctaagctcc ctgagcttga agtagtgcag ttaaatattt ctgcacacat ggattttggc 960

gaagccagac ttgacagcgt taccatcaat ggtaacacat cctattgtgt cactaagcct 1020

tacttcaggc ttgaaactaa ctttatgtgt acaggttgca ctatgaatct gcgcactgat 1080

acctgtagtt ttgacctgtc agcagtaaac aatggcatgt cattctctca attctgtcta 1140

agcactgaat ctggtgcttg tgagatgaaa attattgtta cctacgtatg gaattacttg 1200

ctaaggcagc gtttgtatgt tactgctgta gagggccaga ctcacactgg aaccacttca 1260

gtacatgcaa cagacacttc tagtgtaatc actgatgtct gcactgacta cactatctat 1320

ggagtctcag gtactggcat tattaagcca tcagatctct tattgcacaa tggcatagca 1380

ttcacctctc caacaggtga gctttatgca tttaaaaata taaccactgg caaaaccctt 1440

caggtcttac cgtgtgaaac cccttctcaa ctgattgtga taaacaacac cgttgtcggt 1500

gctatcacat ccagtaattc aactgaaaat aataggttta ctactactat tgtcacacct 1560

actttctttt attccacaaa tgccaccact ttcaactgca ctaagcctgt tttgtcctat 1620

ggacctatca gcgtgtgtag tgatggtgca attgtgggaa catccacatt acagaatact 1680

cgaccatcca tagtttcact atacgatggc gaagttgaaa taccatctgc attttctctt 1740

tccgttcaga cggagtactt gcaagttcaa gcagagcaag ttatagttga ttgtcctcag 1800

tatgtatgca atggcaacag ccgttgtcta caattactgg cacaatacac ctcagcttgc 1860

tctaacattg aagcagctct gcattcctct gcacagttgg atagcagaga gattataaat 1920

atgtttcaaa catcaacaca gtccttgcag ttagctaata ttaccaactt caagggtgac 1980

tacaatttta gcagcttact aaccaccaga attggtggca gatctgctat tgaagacctt 2040

ctttttaata aagttgttac tagtggcctt ggcactgttg atcaggacta caaatcctgc 2100

tctagagaca tggccatcgc tgacttagtt tgttcccagt attacaatgg catcatggtt 2160

ctacctggtg ttgttgatgc tgagaaaatg gcaatgtata ctggctctct tactggagct 2220

atggtatttg gaggactgac tgccgcagcg gcaataccat ttgccacggc agtacaagct 2280

cgcctcaatt atgtcgcact gcaaacaaat gtactacaag aaaaccagaa aattcttgca 2340

gaatcattta accaagcagt tggcaatata tcacttgcac tatcttctgt taatgatgcc 2400

atccagcaaa cttctgaggc tcttaacacc gtagctattg ctattaaaaa gattcaaaca 2460

gttgttaacc agcagggcga ggcattatca cacctgactg cacagctgtc aaacaatttt 2520

caagcaattt cgacttctat tcaagacatt tacaaccgtc ttgaggaagt agaggctaac 2580

cagcaagttg accgtctcat cacaggacgg ttggctgcac ttaatgcata tgttactcag 2640

ttactcaatc agatgtctca gattagacaa tctcgattgt tagctcagca aaagattaat 2700

gagtgtgtca aatcacagtc gtccagatac ggtttctgtg gaaatggcac acacatcttc 2760

tcacttacac agactgcacc aaatggcata tttttcatgc atgcagtgct agtacccaac 2820

aaattcacac gtgtcaacgc ttctgccggc atttgtgtgg ataatacgag aggctactca 2880

ttgcagcctc aacttatact ctaccagttt aataactcct ggagagttac acctagaaat 2940

atgtatgaac ccagactgcc ccggcaggct gatttcatac aattaactga ttgcagcgtt 3000

actttttaca acaccaccgc tgctaatctt cccaatatta tccctgacat tatagatgtc 3060

aatcaaacag tcagtgatat tattgacaat ttacctacag caacacctcc tcagtgggat 3120

gttggtatct ataacaacac tattctcaac ctcaccgttg agattaatga tctacaagag 3180

cggtctaaaa acctctcaca gattgcagat cgtttacaaa attatattga caatcttaac 3240

aatactctag ttgaccttga atggctcaac agagtggaaa cttaccttaa atggccgtgg 3300

tatatatggc ttgccattgc cctggctctt attgcatttg tgacaatcct cataacaatc 3360

tttcttagaa accgcaatcg tcagtactaa 3390

<210> 28

<211> 1129

<212> PRT

<213> Artificial Sequence

<220>

<223> PDCoVS BD Protein - expressed baculodisplay

<400> 28

Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His

1 5 10 15

Ser Ala Phe Ala Asp Asp Leu Leu Asp Leu Leu Thr Phe Pro Gly Ala

20 25 30

His Arg Phe Leu His Lys Pro Thr Arg Asn Ser Ser Ser Leu Tyr Ser

35 40 45

Arg Ala Asn Asn Asn Phe Asp Val Gly Val Leu Pro Gly Tyr Pro Thr

50 55 60

Lys Asn Val Asn Leu Phe Ser Pro Leu Thr Asn Ser Thr Leu Pro Ile

65 70 75 80

Asn Gly Leu His Arg Ser Tyr Gln Pro Leu Met Leu Asn Cys Leu Thr

85 90 95

Lys Ile Thr Asn His Thr Leu Ser Met Tyr Leu Leu Pro Ser Glu Ile

100 105 110

Gln Thr Tyr Ser Cys Gly Gly Ala Met Val Lys Tyr Gln Thr His Asp

115 120 125

Ala Val Arg Ile Ile Leu Asp Leu Thr Ala Thr Asp His Ile Ser Val

130 135 140

Glu Val Val Gly Gln His Gly Glu Asn Tyr Val Phe Val Cys Ser Glu

145 150 155 160

Gln Phe Asn Tyr Thr Thr Ala Leu His Asn Ser Thr Phe Phe Ser Leu

165 170 175

Asn Ser Glu Leu Tyr Cys Phe Thr Asn Asn Thr Tyr Leu Gly Ile Leu

180 185 190

Pro Pro Asp Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr

195 200 205

Ala Asn Gly Tyr Leu Leu Gly Thr Leu Pro Ile Thr Val Asn Tyr Val

210 215 220

Arg Leu Tyr Arg Gly His Leu Ser Ala Asn Ser Ala Tyr Phe Ala Leu

225 230 235 240

Ala Asn Leu Thr Asp Thr Leu Ile Thr Leu Thr Asn Thr Thr Ile Ser

245 250 255

Gln Ile Thr Tyr Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln

260 265 270

Arg Ser Ser His Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser

275 280 285

Ala Val Arg Ala Arg Gln Arg Thr Ile Val Thr Leu Pro Lys Leu Pro

290 295 300

Glu Leu Glu Val Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly

305 310 315 320

Glu Ala Arg Leu Asp Ser Val Thr Ile Asn Gly Asn Thr Ser Tyr Cys

325 330 335

Val Thr Lys Pro Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly

340 345 350

Cys Thr Met Asn Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala

355 360 365

Val Asn Asn Gly Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ser

370 375 380

Gly Ala Cys Glu Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu

385 390 395 400

Leu Arg Gln Arg Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr

405 410 415

Gly Thr Thr Ser Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp

420 425 430

Val Cys Thr Asp Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile

435 440 445

Lys Pro Ser Asp Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro

450 455 460

Thr Gly Glu Leu Tyr Ala Phe Lys Asn Ile Thr Thr Gly Lys Thr Leu

465 470 475 480

Gln Val Leu Pro Cys Glu Thr Pro Ser Gln Leu Ile Val Ile Asn Asn

485 490 495

Thr Val Val Gly Ala Ile Thr Ser Ser Asn Ser Thr Glu Asn Asn Arg

500 505 510

Phe Thr Thr Thr Ile Val Thr Pro Thr Phe Phe Tyr Ser Thr Asn Ala

515 520 525

Thr Thr Phe Asn Cys Thr Lys Pro Val Leu Ser Tyr Gly Pro Ile Ser

530 535 540

Val Cys Ser Asp Gly Ala Ile Val Gly Thr Ser Thr Leu Gln Asn Thr

545 550 555 560

Arg Pro Ser Ile Val Ser Leu Tyr Asp Gly Glu Val Glu Ile Pro Ser

565 570 575

Ala Phe Ser Leu Ser Val Gln Thr Glu Tyr Leu Gln Val Gln Ala Glu

580 585 590

Gln Val Ile Val Asp Cys Pro Gln Tyr Val Cys Asn Gly Asn Ser Arg

595 600 605

Cys Leu Gln Leu Leu Ala Gln Tyr Thr Ser Ala Cys Ser Asn Ile Glu

610 615 620

Ala Ala Leu His Ser Ser Ala Gln Leu Asp Ser Arg Glu Ile Ile Asn

625 630 635 640

Met Phe Gln Thr Ser Thr Gln Ser Leu Gln Leu Ala Asn Ile Thr Asn

645 650 655

Phe Lys Gly Asp Tyr Asn Phe Ser Ser Leu Leu Thr Thr Arg Ile Gly

660 665 670

Gly Arg Ser Ala Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Ser

675 680 685

Gly Leu Gly Thr Val Asp Gln Asp Tyr Lys Ser Cys Ser Arg Asp Met

690 695 700

Ala Ile Ala Asp Leu Val Cys Ser Gln Tyr Tyr Asn Gly Ile Met Val

705 710 715 720

Leu Pro Gly Val Val Asp Ala Glu Lys Met Ala Met Tyr Thr Gly Ser

725 730 735

Leu Thr Gly Ala Met Val Phe Gly Gly Leu Thr Ala Ala Ala Ala Ile

740 745 750

Pro Phe Ala Thr Ala Val Gln Ala Arg Leu Asn Tyr Val Ala Leu Gln

755 760 765

Thr Asn Val Leu Gln Glu Asn Gln Lys Ile Leu Ala Glu Ser Phe Asn

770 775 780

Gln Ala Val Gly Asn Ile Ser Leu Ala Leu Ser Ser Val Asn Asp Ala

785 790 795 800

Ile Gln Gln Thr Ser Glu Ala Leu Asn Thr Val Ala Ile Ala Ile Lys

805 810 815

Lys Ile Gln Thr Val Val Asn Gln Gln Gly Glu Ala Leu Ser His Leu

820 825 830

Thr Ala Gln Leu Ser Asn Asn Phe Gln Ala Ile Ser Thr Ser Ile Gln

835 840 845

Asp Ile Tyr Asn Arg Leu Glu Glu Val Glu Ala Asn Gln Gln Val Asp

850 855 860

Arg Leu Ile Thr Gly Arg Leu Ala Ala Leu Asn Ala Tyr Val Thr Gln

865 870 875 880

Leu Leu Asn Gln Met Ser Gln Ile Arg Gln Ser Arg Leu Leu Ala Gln

885 890 895

Gln Lys Ile Asn Glu Cys Val Lys Ser Gln Ser Ser Arg Tyr Gly Phe

900 905 910

Cys Gly Asn Gly Thr His Ile Phe Ser Leu Thr Gln Thr Ala Pro Asn

915 920 925

Gly Ile Phe Phe Met His Ala Val Leu Val Pro Asn Lys Phe Thr Arg

930 935 940

Val Asn Ala Ser Ala Gly Ile Cys Val Asp Asn Thr Arg Gly Tyr Ser

945 950 955 960

Leu Gln Pro Gln Leu Ile Leu Tyr Gln Phe Asn Asn Ser Trp Arg Val

965 970 975

Thr Pro Arg Asn Met Tyr Glu Pro Arg Leu Pro Arg Gln Ala Asp Phe

980 985 990

Ile Gln Leu Thr Asp Cys Ser Val Thr Phe Tyr Asn Thr Thr Ala Ala

995 1000 1005

Asn Leu Pro Asn Ile Ile Pro Asp Ile Ile Asp Val Asn Gln Thr

1010 1015 1020

Val Ser Asp Ile Ile Asp Asn Leu Pro Thr Ala Thr Pro Pro Gln

1025 1030 1035

Trp Asp Val Gly Ile Tyr Asn Asn Thr Ile Leu Asn Leu Thr Val

1040 1045 1050

Glu Ile Asn Asp Leu Gln Glu Arg Ser Lys Asn Leu Ser Gln Ile

1055 1060 1065

Ala Asp Arg Leu Gln Asn Tyr Ile Asp Asn Leu Asn Asn Thr Leu

1070 1075 1080

Val Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr Leu Lys Trp

1085 1090 1095

Pro Trp Tyr Ile Trp Leu Ala Ile Ala Leu Ala Leu Ile Ala Phe

1100 1105 1110

Val Thr Ile Leu Ile Thr Ile Phe Leu Arg Asn Arg Asn Arg Gln

1115 1120 1125

Tyr

<210> 29

<211> 27960

<212> DNA

<213> Artificial Sequence

<220>

<223> Complete Genome Nucleic Acid (cDNA) Sequence equivalent of the

(+) single-stranded RNA genome of PEDV Isolate 1251-125-10

(125-10)

<400> 29

agactcttgt ctactcaatt caactaaacg aaattttgtc cttccggccg catgtccatg 60

ctgctggaag ctgacgtgga atttcattag gtttgcttaa gtagccatcg caagtgctgt 120

gctgtcctct agttcctggt tggcgttccg tcgccttcta catactagac aaacagcctt 180

cctccggttc cgtctggggg ttgtgtggat aactagttcc gtctagtttg aaaccagtaa 240

ctgtcggcta tggctagcaa ccatgttaca ttggcttttg ccaatgatgc agaaatttca 300

gcttttggct tttgcactgc tagtgaagcc gtctcatact attctgaggc cgccgctagt 360

ggatttatgc aatgccgttt cgtgtccttc gatctcgctg acactgttga gggattgctt 420

cccgaagact atgtcatggt ggtggtcggc actaccaagc ttagtgcgta tgtggacact 480

tttggtagcc gccccaaaaa catttgtggt tggctgttat tttctaactg taattacttc 540

ctcgaagagt tagagcttac ttttggtcgt cgtggtggta acatcgtgcc agttgaccaa 600

tacatgtgtg gcgctgacgg taaacctgtt cttcaggaat ccgaatggga gtatacagat 660

ttctttgctg actccgaaga cggtcaactc aacattgctg gtatcactta tgtgaaggcc 720

tggattgtag agcgatcgga tgtctcttat gcgagtcaga atttaacatc tattaagtct 780

attacttact gttcaaccta tgagcatact tttcctgatg gtactgccat gaaggttgca 840

cgtactccaa agattaagaa gactgttgtc ttgtctgagc cacttgctac tatctacagg 900

gaaattggtt ctccttttgt ggataatggg agcgatgctc gttctatcat taagagacca 960

gtgttcctcc acgcttttgt taagtgtaag tgtggtagtt atcattggac tgttggtgat 1020

tggacttcct atgtctccac ttgctgtggc tttaagtgta agccagtcct tgtggcttca 1080

tgctctgcta cgcctggttc tgttgtggtt acgcgcgctg gtgctggcac tggtgttaag 1140

tattacaaca acatgttcct gcgccatgtg gcagacattg atgggttggc attctggcga 1200

attctcaagg tgcagtccaa agacgacctc gcttgctctg gtaaattcct tgaacaccat 1260

gaggaaggtt tcacagatcc ttgctacttt ttgaatgact cgagcattgc tactaagctc 1320

aagtttgaca tccttagtgg caagttttct gatgaagtca aacaagctat ctttgctggt 1380

catgttgttg ttggcagcgc gctcgttgac attgttgacg atgcactggg acagccttgg 1440

tttatacgta agcttggtga ccttgcaagt gcagcttggg agcagcttaa ggctgtcgtt 1500

agaggcctta acctcctgtc tgatgaggtc gtgctctttg gcaaaagact tagctgtgcc 1560

actcttagta tcgttaacgg tgtttttgag ttcatcgccg aagtgcctga gaagttggct 1620

gcggctgtta cagtttttgt caacttcttg aatgagcttt ttgagtctgc ctgtgactgc 1680

ttaaaggtcg gaggtaaaac ctttaacaag gttggctctt atgttctttt tgacaacgca 1740

ttggttaagc ttgtcaaggc aaaagttcgc ggcccacgac aggcaggtgt ttgtgaagtt 1800

cgttacacaa gccttgttat tgggagtact accaaggtgg tttccaagcg cgttgaaaat 1860

gccaatgtga atctcgtcgt cgttgacgag gatgtgaccc tcaacaccac tggtcgtaca 1920

gttgttgttg acggacttgc attcttcgag agtgacgggt tttacagaca tcttgctgat 1980

gctgacgttg tcattgaaca tcctgtttat aagtctgctt gtgagctcaa gccagttttt 2040

gagtgtgacc caatacctga ttttcctatg cctgtggccg ctagtgttgc agagctttgt 2100

gtgcaaactg atctgttgct taaaaattac aacactcctt ataaaactta cagctgcgtt 2160

gtgagaggtg ataagtgttg tatcacttgc accttacatt tcacagcacc aagttatatg 2220

gaggctgctg ctaattttgt agacctctgt accaagaaca ttggtactgc tggttttcat 2280

gagttttaca ttacggccca tgaacaacag gatctgcaag ggttcgtaac cacttgttgc 2340

acgatgtcag gttttgagtg ttttatgcct ataatcccac agtgtccagc agtgcttgaa 2400

gagattgatg gtggtagcat ctggcggtct tttatcactg gtcttaatac aatgtgggat 2460

ttttgcaagc atcttaaagt cagctttgga ctagatggca ttgttgtcac tgtagcacgc 2520

aaatttaaac gacttggtgc tctcttggca gaaatgtata acacttacct ttcaactgtg 2580

gtggaaaact tggtactggc cggtgttagc ttcaagtatt atgccaccag tgtcccaaaa 2640

attgttttgg gctgttgttt tcacagtgtt aaaagtgttc ttgcaagtgc cttccagatt 2700

cctgtccagg caggcgttga gaagtttaaa gtcttcctta actgtgttca ccctgttgta 2760

ccacgtgtca ttgaaacttc ttttgtggaa ttagaagaga cgacatttaa accaccagca 2820

ctcaatggta gtattgctat tgttgatggc tttgctttct attatgatgg aacactatac 2880

tatcccaccg atggtaatag cgttgttcct atctgcttta agaagaaagg tggtggtgat 2940

gtcaaattct ctgatgaagt ctctgttaaa accattgacc cagtttataa ggtctccctt 3000

gaatttgagt tcgagtctga gactattatg gctgtgctta ataaggctgt tggtaattgt 3060

atcaaggtta caggtggttg ggacgatgtt gttgagtata tcaatgttgc cattgaggtt 3120

cttaaagatc acatcgatgt gcctaagtac tacatctatg atgaggaagg tggcaccgat 3180

cctaatctgc ccgtaatggt ttctcagtgg ccgttgaatg atgacacgat ctcacaggat 3240

ctgcttgatg ttgaagttgt tactgatgcg ccagttgatt tcgagggtga tgaagtagac 3300

tcctctgacc ctgataaggt ggcagacgtg gctaactctg agcctgagga tgacggtctt 3360

aatgtagctc ctgaaacaaa tgtagagtct gaagttgagg aagttgccgc aaccttgtcc 3420

tttattaaag atacaccttc cacagttact aaggatcctt ttgcttttga ctttgcaagc 3480

tatggaggac ttaaggtttt aagacaatct cataacaact gctgggttac ttctaccttg 3540

gtgcagctac aattgcttgg catcgttgat gaccctgcaa tggagctttt tagtgctggt 3600

agagttggtc caatggttcg caaatgctat gagtcacaaa aggctatctt gggatctttg 3660

ggtgatgtgt cggcttgcct agagtctctg actaaggacc tacacacact taagattacc 3720

tgttctgtag tctgtggttg tggtactggt gaacgtatct atgatggttg tgcttttcgt 3780

atgacgccaa ctttggaacc gttcccatat ggtgcttgtg ctcagtgtgc tcaagttttg 3840

atgcacactt ttaaaagtat tgttggcacc ggcatctttt gtcgagatac tactgctctc 3900

tccttggatt ctttggttgt aaaacctctt tgtgcggctg cttttatagg caaggatagt 3960

ggtcattatg tcactaactt ttatgatgct gctatggcta ttgatggtta tggtcgtcat 4020

cagataaagt atgacacact gaacactatt tgtgttaaag acgttaattg gacagcacct 4080

tttgtcccag acgttgagcc tgtattggag cctgttgtca aacctttcta ttcttataag 4140

aatgttgatt tttaccaagg agattttagt gaccttgtta aacttccatg tgattttgtt 4200

gttaatgctg caaatgagaa tttgtctcac ggtggcggca tagcaaaggc cattgatgtt 4260

tataccaagg gcatgttgca gaagtgctcg aatgattaca ttaaagcaca cggtcccatt 4320

aaagttggac gtggtgtcat gttggaggca ttaggtctta aggtctttaa tgttgttggt 4380

ccacgtaagg gtaagcatgc acctgagctt cttgttaagg cttataagtc cgtttttgct 4440

aattcaggtg ttgctcttac acctttgatt agtgttggaa tttttagtgt tcctttggaa 4500

gaatctttat ctgcttttct tgcatgtgtt ggtgatcgcc actgtaagtg cttttgttat 4560

agtgacaaag agcgcgaggc gatcattaat tacatggatg gcttggtaga tgctattttc 4620

aaagatgcac ttgttgatac tactcctgtc caggaagatg ttcaacaagt ttcacaaaaa 4680

ccagttttgc ctaattttga acctttcagg attgaaggtg ctcatgcttt ctatgagtgc 4740

aaccctgaag gtttgatgtc attaggtgct gacaagctgg tgttgtttac aaattccaat 4800

ttggattttt gtagcgttgg taagtgtctt aacaatgtga ctggcggtgc attgcttgaa 4860

gccataaatg tatttaaaaa gagtaacaaa acagtgcctg ctggcaactg tgttactttt 4920

gagtgtgcag atatgatttc tattactatg gtagtattgc catctgacgg tgatgctaat 4980

tatgacaaaa attatgcacg cgccgtcgtc aaggtatcta agcttaaagg caagttattg 5040

cttgctgttg gtgatgccat gttgtattcc aagttgtccc acctcagcgt gttaggtttc 5100

gtatccacac ctgatgatgt ggagcgtttc tacgcaaata agagtgtggt tattaaagtt 5160

actgaggata cacgtagtgt taagactgtt aaagtagaat ccactgttac ttatggacaa 5220

caaattggac cttgtcttgt taatgacacc gttgtcacag acaacaaacc tgttgttgct 5280

gatgttgtag ctaaggttgt accaagtgct aattgggatt cacattatgg ttttgataag 5340

gctggtgagt tccacatgct agaccatact gggtttgcct ttcctagtga agttgttaac 5400

ggtaggcgtg tgcttaaaac cacagataat aactgttggg ttaatgttac atgtttacaa 5460

ttacagtttg ctagatttag gttcaagtca gcaggtctac aggctatgtg ggagtcctat 5520

tgtactggtg atgttgctat gtttgtgcat tggttgtact ggcttactgg tgttgacaaa 5580

ggtcagccta gtgattcaga aaatgcactt aacatgttgt ctaagtacat tgttcctgct 5640

ggttctgtca ctattgaacg tgtcacgcat gacggttgtt gttgtagtaa gcgtgttgtc 5700

actgcaccag ttgtgaatgc tagcgtgttg aagcttggcg tcgaggatgg tctttgtcca 5760

catggtctta actacattga caaagttgtt gtagttaaag gtactacaat tgttgtcaat 5820

gttggaaaac ctgtagtggc accatcgcac ctctttctta agggtgtttc ctacacaaca 5880

ttcctagata atggtaacgg tgttgccggc cattatactg tttttgatca tgacactggt 5940

atggtgcatg atggagatgt ttttgtacca ggtgatctca atgtgtctcc tgttacaaat 6000

gttgtcgtct cagagcagac ggctgttgtg attaaagacc ctgtgaagaa agtagagtta 6060

gacgctacaa agctgttaga cactatgaat tatgcatcgg aaagattctt ttcctttggt 6120

gattttatgt cacgtaattt aattacagtg tttttgtaca tccttagtat tttgggtctc 6180

tgttttaggg cctttcgtaa gagggatgtt aaagttctag ctggtgtacc ccaacgtact 6240

ggtattatat tgcgtaaaag tgtgcgctat aatgcaaagg ctttgggtgt cttcttcaag 6300

ctaaaacttt attggttcaa agttcttggt aagtttagtt tgggtattta tgcattgtat 6360

gcattactat tcatgacaat acgctttaca cctataggtg gccctgtttg tgatgatgtt 6420

gttgctggtt atgctaattc tagttttgac aagaatgagt attgcaacag tgttatttgt 6480

aaggtctgtc tctatgggta ccaggaactt tcggacttct ctcacacaca ggtagtatgg 6540

caacacctta gagacccatt aattggtaat gtgatgcctt tcttttattt ggcatttctg 6600

gcaatttttg ggggtgttta tgtaaaggct attactctct attttatttt ccagtatctt 6660

aacatacttg gtgtgttttt gggcctacaa cagtccattt ggtttttgca gcttgtgcct 6720

tttgatgtct ttggtgacga gatcgtcgtc tttttcatcg ttacacgcgt attgatgttc 6780

cttaagcatg ttttccttgg ctgcgataag gcatcttgtg tggcttgctc taagagtgct 6840

cgccttaagc gcgttcctgt ccagactatt tttcagggta ctagcaaatc cttctacgta 6900

catgccaatg gtggttctaa gttctgtaag aagcacaatt tcttttgttt aaattgtgat 6960

tcttatggtc caggctgcac ttttattaat gacgtcattg caactgaagt tggtaatgtt 7020

gtcaaactta atgtgcaacc gacaggtcct gccactattc ttattgacaa ggttgaattc 7080

agtaatggtt tttactatct ttatagtggt gacacatttt ggaagtacaa ctttgacata 7140

acagataaca aatacacttg caaagagtca cttaaaaatt gtagcataat cacagacttt 7200

attgttttta acaataatgg ttccaatgta aatcaggtta agaatgcatg tgtgtatttt 7260

tcacagatgc tttgtaaacc tgttaagtta gtggactcag cgttgttggc cagtttgtct 7320

gttgattttg gtgcaagctt acatagtgct tttgttagtg tgttgtcgaa tagttttggc 7380

aaagacctgt caagttgtaa tgacatgcag gattgcaaga gcacattggg ttttgatgat 7440

gtaccattgg atacctttaa tgctgctgtt gctgaggctc atcgttacga tgtcctcttg 7500

actgacatgt cgttcaacaa ttttaccacc agttatgcaa aaccagagga aaaacttccc 7560

gtccatgaca ttgccacgtg tatgcgtgta ggtgccaaga ttgttaatca taacgttctt 7620

gtcaaggata gtatacctgt ggtgtggctt gtacgtgatt tcattgccct ttctgaagaa 7680

actaggaagt acattattcg tacgactaaa gttaagggta taaccttcat gttgaccttt 7740

aatgattgtc gtatgcatac taccatacct actgtttgca ttgcaaataa gaagggtgca 7800

ggtcttccta gtttttcaaa ggttaagaaa ttcttctggt ttttgtgtct gttcatagtt 7860

gctgttttct ttgcactaag cttttttgat tttagtactc aggttagcag tgatagtgat 7920

tatgacttca agtatattga gagtggccag ttgaagactt ttgacaatcc acttagttgt 7980

gtgcataatg tctttagtaa cttcgaccag tggcatgatg ccaagtttgg tttcaccccc 8040

gtcaacaatc ctagttgtcc tatagtcgtt ggtgtatcag acgaagcgcg cactgttcca 8100

ggtatcccag caggtgttta tttagctggt aaaacacttg tttttgctat taacaccatt 8160

tttggtacat ctggtttgtg ctttgatgct agtggcgttg ctgataaggg cgcttgcatt 8220

tttaattcgg cttgcaccac attatctggt ttgggtggaa ctgctgtcta ctgttataag 8280

aatggtctag ttgaaggtgc taaactttat agtgagttgg cacctcatag ctactataaa 8340

atggtagatg gtaatgctgt gtctttacct gaaattatct cacgcggctt tggcatccgt 8400

actatccgta caaaggctat gacctactgt cgcgttggcc agtgtgtgca atctgcagaa 8460

ggtgtttgtt ttggcgccga tagattcttt gtctataatg cagaatctgg ttctgacttt 8520

gtttgtggca cagggctctt tacattgttg atgaacgtta ttagtgtttt ttccaagaca 8580

gtaccagtaa ctgtgttgtc tggtcaaata ctttttaatt gcattattgc ttttgctgct 8640

gttgcggtgt gtttcttatt tacaaagttt aagcgcatgt tcggtgatat gtctgttggc 8700

gttttcactg tcggtgcttg tactttgttg aacaatgttt cctacattgt aacacagaac 8760

acacttggca tgttgggcta tgcaactttg tactttttgt gcactaaagg tgttagatat 8820

atgtggattt ggcatttggg atttttgatc tcatatatac ttattgcacc atggtgggtt 8880

ttgatggttt atgccttttc agccattttt gagtttatgc ctaacctttt taagcttaag 8940

gtttcaacac aactttttga gggtgacaag ttcgtaggct cttttgaaaa tgctgcagca 9000

ggtacatttg tgcttgatat gcatgcctat gagagacttg ccaactctat ctcaactgaa 9060

aaactgcgtc agtatgctag tacttacaat aagtacaagt attattcagg cagtgcttca 9120

gaggctgatt acaggcttgc ttgttttgcc catttggcca aggctatgat ggattatgct 9180

tctaatcaca acgacacgtt atacacacca cccactgtga gttacaattc aactctacag 9240

gctggcttgc gtaagatggc acaaccatct ggtgttgttg agaagtgcat agttcgtgtt 9300

tgctatggta atatggctct taatggccta tggcttggtg atactgttat ctgcccacgc 9360

catgttatag cgtctagtac tactagcact atagattatg actatgccct ttctgtttta 9420

cgcctccaca acttctccat ttcatctggt aatgttttcc taggtgttgt gggtgtaacc 9480

atgcgaggtg ctttgttgca gataaaggtt aatcaaaaca atgtccacac gcctaagtac 9540

acctatcgca cagttagacc gggtgaatct tttaatatct tggcgtgcta tgatggttct 9600

gcagctggtg tttacggcgt taacatgcgc tctaattaca ctattagagg ctcgttcatt 9660

aatggcgctt gtggttcacc tggttataac attaacaatg gtaccgttga gttttgctat 9720

ttacaccagc ttgaacttgg ttcaggctgt catgttggta gcgacttaga tggtgttatg 9780

tatggtggtt atgaggacca acctactttg caagttgaag gcgctagtag tctgtttaca 9840

gagaatgtgt tggcatttct ttatgcagca ctcattaatg gttctacctg gtggcttagt 9900

tcttctagga ttgctgtaga caggtttaat gagtgggctg ttcataatgg tatgacaaca 9960

gtagttaata ctgattgctt ttctattctt gctgctaaga ctggtgttga tgtacaacgt 10020

ttgttggcct caatccagtc tctgcataag aattttggtg gaaagcaaat tcttggctat 10080

acctcgttga cagatgagtt tactacaggt gaagttatac gtcaaatgta tggcgttaat 10140

cttcagagtg gttatgtttc acgcgcctgt agaaatgtct tgctggttgg ttcttttctg 10200

actttctttt ggtcagaatt agtttcctac actaagttct tttgggtaaa tcctggttat 10260

gtcacaccta tgtttgcgtg tttgtcattg ctgtcctcac ttttgatgtt cacactcaag 10320

cataagacat tgtttttcca ggtctttcta atacctgctc tgattgttac atcttgcatt 10380

aatttggcat ttgatgttga agtctacaac tatttggcag agcattttga ttaccatgtt 10440

tctctcatgg gttttaatgc acaaggtctt gttaacatct ttgtctgctt tgttgttacc 10500

attttacacg gcacatacac atggcgcttt tttaacacac ctgtgagttc tgtcacttat 10560

gtggtagctt tgctgactgc ggcatataac tatttttacg ctagtgacat tcttagttgt 10620

gctatgacac tatttgctag tgtgactggc aactggttcg ttggtgctgt ttgttataaa 10680

gctgctgttt atatggcctt gagatttcct acttttgtgg ctatttttgg tgatattaag 10740

agtgttatgt tctgttacct tgtgttgggt tattttacct gttgcttcta cggtattctc 10800

tactggttca acaggttttt taaggttagt gtaggtgtct atgactatac tgttagtgct 10860

gctgagttta agtatatggt tgctaacggc ctacgtgcac caactggaac acttgattca 10920

ctacttctgt ctgccaaatt gattggtatt ggtggtgagc ggaatattaa gatttcttcc 10980

gttcagtcta aactgactga tattaagtgt agtaacgttg tgcttttagg ctgtctctct 11040

agcatgaatg tctcagcaaa ttcaacagaa tgggcctatt gtgttgactt gcataacaag 11100

atcaacttgt gtaatgaccc agaaaaagcg caggaaatgc tacttgcttt gttggcattt 11160

ttccttagta agaatagtgc ttttggttta gatgacttat tggaatccta ttttaatgac 11220

aatagtatgt tgcagagtgt tgcatctact tatgtcggtt tgccttctta tgtcatttat 11280

gaaaatgcac gccaacagta tgaagatgct gttaataatg gttctccacc tcagttggtt 11340

aagcaattgc gccatgccat gaatgtagca aagagcgaat ttgaccgtga ggcttctact 11400

cagcgtaagc ttgatagaat ggcggaacag gctgcagcac agatgtacaa agaggcacga 11460

gcagttaata ggaagtccaa agttgtaagt gctatgcatt cactgctttt tggtatgttg 11520

agacgtttgg acatgtcttc tgtagacacc attctcaact tggcaaagga tggggttgta 11580

cctctgtctg tcataccggc agtcagtgct actaagctta acattgttac ttctgatatc 11640

gattcttata atcgtatcca gcgtgaggga tgtgtccact acgctggtac catttggaat 11700

ataattgata tcaaggacaa tgatggcaag gtggtacacg ttaaggaggt aaccgcacag 11760

aatgctgagt ccctgtcatg gcccctggtc cttgggtgtg agcgtattgt caagctccag 11820

aataatgaaa ttattcctgg taagctgaag cagcgctcca ttaaggcaga aggagatggc 11880

atagttggag aaggtaaggc actttacaat aatgagggtg gacgtacttt tatgtatgct 11940

ttcatctcgg acaaaccgga cctgcgtgta gtcaagtggg agttcgatgg tggttgtaac 12000

actattgagc tagaaccacc acgtaagttc ttggtggatt ctcctaatgg tgcacagatc 12060

aagtatctct actttgttcg taaccttaac acgttacgta ggggtgctgt tctcggctac 12120

ataggtgcca ctgtacgctt gcaggctggt aaacaaacag aacaggctat taactcttca 12180

ttgttgacac tttgcgcttt cgctgtggat cctgctaaga cctacatcga tgctgtcaaa 12240

agtggtcaca aaccagtagg taactgtgtt aagatgttgg ccaatggttc tggtaatgga 12300

caagctgtta ctaatggtgt ggaggctagt actaaccagg attcatacgg tggtgcgtcc 12360

gtgtgtctat attgtagagc acatgttgag catccatcta tggatggttt ttgcagactg 12420

aaaggcaagt acgtacaggt tccactaggt acagtggatc ctatacgttt tgtacttgag 12480

aatgacgttt gcaaggtttg tggttgttgg ctggctaatg gctgcacttg tgacagatcc 12540

attatgcaaa gcactgatat ggcttattta aacgagtacg gggctctagt gcagctcgac 12600

tagagccctg taacggtact gatacacaac atgtgtatcg tgcttttgac atctacaaca 12660

aggatgttgc ttgtctaggt aaattcctca aggtgaactg tgttcgcctg aagaatttgg 12720

ataagcatga tgcattctat gttgtcaaaa gatgtaccaa gtctgcgatg gaacacgagc 12780

aatccatcta tagcagactt gaaaagtgtg gagccgtagc cgaacacgat ttcttcactt 12840

ggaaggatgg tcgtgccatc tatggtaacg tttgtagaaa ggatcttacc gagtatacta 12900

tgatggattt gtgttacgct ttacgtaact ttgatgaaaa caattgcgat gttcttaaga 12960

gcattttaat taaggtaggc gcttgtgagg agtcctactt caataataaa gtctggtttg 13020

accctgttga aaatgaagac attcatcgtg tctatgcatt gttaggtacc attgtttcac 13080

gtgctatgct taaatgcgtt aagttctgtg atgcaatggt tgaacaaggt atagttggtg 13140

ttgtcacatt agataatcag gatcttaatg gtgattttta tgattttggt gattttactt 13200

gtagcatcaa gggaatgggt atacccattt gcacatcata ttactcttat atgatgcctg 13260

ttatgggtat gactaattgc cttgctagtg agtgttttgt taagagtgat atatttggtg 13320

aggatttcaa gtcatatgac ctgctggaat atgatttcac ggagcataag acagcactct 13380

tcaacaagta tttcaagtat tggggactgc aataccaccc taactgtgtg gactgcagtg 13440

atgagcagtg catagttcac tgtgccaact tcaatacgtt gttttccact actataccta 13500

ttacggcatt tggacctttg tgtcgcaagt gttggattga tggtgttcca ctggtaacta 13560

cagctggtta tcattttaaa cagttaggta tagtttggaa caatgacctc aacttacact 13620

ctagcaggct ctctattaac gaattactcc agttttgtag tgatcctgca ttgcttatag 13680

catcatcacc agcccttgtt gatcagcgta ctgtttgctt ttcagttgca gcgctaggta 13740

caggtatgac taaccagact gttaaacctg gccatttcaa taaggagttt tatgacttct 13800

tacttgagca aggtttcttt tctgagggct ctgagcttac tttaaagcac ttcttctttg 13860

cacagaaggg tgatgcagct gttaaggatt ttgactacta taggtataat agacctactg 13920

ttctggacat ttgccaagct cgcgtcgtgt atcaaatagt gcaacgctat tttgatattt 13980

acgaaggtgg ttgtatcact gctaaagagg tggttgttac aaaccttaac aagagcgcag 14040

gttatccttt gaacaagttt ggtaaagctg gtctttacta tgagtcttta tcctatgagg 14100

aacaggatga actttatgct tatactaagc gtaacatcct gcccactatg acacagctca 14160

accttaaata tgctataagt ggcaaagaac gtgcacgcac agtgggtggt gtttcgcttt 14220

tgtcaaccat gactactcgg cagtatcatc agaaacacct taagtccata gttaatacta 14280

ggggcgcttc ggttgttatt ggtactacta agttttatgg tggttgggac aatatgctta 14340

agaaccttat tgatggtgtt gaaaatccgt gtcttatggg ttgggactac ccaaagtgcg 14400

acagagcact gcccaatatg atacgtatga tttcagccat gattttaggc tctaagcaca 14460

ccacatgctg cagttccact gaccgctttt tcaggttgtg caatgaattg gctcaagtcc 14520

ttactgaggt tgtttattct aatggaggtt tttatttgaa gccaggtggt actacctctg 14580

gtgatgcaac caccgcatat gcaaactcag tttttaatat cttccaagca gtaagtgcca 14640

atgttaacaa acttcttagt gttgacagca atgtctgtca taatttagaa gttaagcaat 14700

tgcagcgtaa gctttatgag tgctgttata gatcaactac cgtcgatgac cagttcgtcg 14760

ttgagtatta tggttacttg cgtaaacatt tttcaatgat gattctttct gatgatggcg 14820

ttgtttgtta taacaatgac tatgcatcac ttggttatgt cgctgatctt aacgcattca 14880

aggctgtttt gtattaccag aacaatgtct tcatgagcgc ctctaaatgt tggatcgagc 14940

ctgacattaa taaaggtcct catgaatttt gctcgcagca tactatgcag attgtcgata 15000

aagatggtac ttattacctt ccttaccctg atccttcaag aattctctct gcaggtgtgt 15060

ttgttgatga cgttgttaaa actgatgcag ttgtattgct tgaacgttat gtgtcattgg 15120

ctatagatgc ctacccgtta tctaagcatg aaaaccctga atataagaag gtgttttatg 15180

tgcttttgga ttgggttaag catctgtaca aaactcttaa tgctggtgtg ttagagtctt 15240

tttctgtcac acttttggaa gattctactg ctaaattctg ggatgagagc ttttatgcca 15300

acatgtatga gaaatctgca gttttacaat ctgcagggct ttgtgttgtt tgtggctctc 15360

aaactgtttt acgttgtggt gattgtctac ggcgtcctat gctttgtact aagtgtgctt 15420

atgatcatgt cattggaaca actcacaagt tcattttggc catcactcca tatgtgtgtt 15480

gtgcttcaga ttgtggtgtc aatgatgtaa ctaagctcta cttaggtggt cttagttatt 15540

ggtgtcatga ccacaagcca cgtcttgcat tcccgttgtg ctctgctggt aatgtttttg 15600

gcttgtacaa aaattctgct accggctcac ccgatgttga agactttaat cgcattgcta 15660

catccgattg gactgatgtt tctgactaca ggttggcaaa tgatgtcaag gactcattgc 15720

gtctgtttgc agcggaaact atcaaggcca aggaggagag cgttaagtca tcctatgctt 15780

gtgcaacact acatgaggtt gtaggaccta aagagttgtt gctcaaatgg gaagtcggca 15840

gacccaaacc accccttaat agaaattcgg ttttcacttg ttatcatata acgaagaaca 15900

ccaaatttca aatcggtgag tttgtgtttg agaaggcaga atatgataat gatgctgtaa 15960

catataaaac taccgccaca acaaaacttg ttcctggcat ggtttttgtg cttacctcac 16020

ataatgttca gccattgcgc gcaccgacca ttgctaatca agaacgttat tccactatac 16080

ataagttgca tcctgctttt aacatacctg aagcttattc tagcttagtg ccctattacc 16140

aattgattgg taagcagaag attacaacta ttcagggacc tcccggtagt ggtaaatctc 16200

actgtgttat agggctaggt ttgtactatc caggtgcacg tatagtgttt acagcttgtt 16260

ctcatgcagc ggtcgattca ctttgtgtga aagcttccac tgcttatagc aatgacaaat 16320

gttcacgcat cataccacag cgcgctcgtg ttgagtgtta tgatggtttc aagtctaata 16380

atactagtgc tcagtacctt ttctctactg tcaatgcttt gccagagtgc aatgcggaca 16440

ttgttgtggt ggatgaggtc tctatgtgca ctaattatga cttgtctgtc ataaatcagc 16500

gcatcagcta taggcatgta gtctatgttg gtgaccctca acagctgcct gcaccacgtg 16560

ttatgatttc acgtggtact ttggaaccaa aggactacaa cgttgtcact caacgcatgt 16620

gtgcccttaa gcctgatgtt ttcttgcaca agtgttatcg ctgtcctgct gagatagtgc 16680

gtactgtgtc tgagatggtc tatgaaaacc aattcattcc tgtgcaccca gatagcaagc 16740

agtgttttaa aatcttttgc aagggtaatg ttcaggttga taatggttca agcattaatc 16800

gcaggcaatt ggatgttgtg cgtatgtttt tggctaaaaa tcctaggtgg tcaaaggctg 16860

tttttatttc tccttataac agccagaatt atgttgccag ccgcatgcta ggtctacaaa 16920

ttcagacagt tgactcatcc cagggtagtg agtatgacta tgtcatttac acacaaactt 16980

cagatactgc ccatgcctgt aatgttaaca ggtttaatgt tgccatcaca agggccaaga 17040

aaggcatatt atgtataatg tgcgataggt ccctttttga tgtgcttaaa ttctttgagc 17100

ttaaattgtc tgatttgcag gctaatgagg gttgtggtct ttttaaagac tgtagcagag 17160

gtgatgatct gttgccacca tctcacgcta acaccttcat gtctttagcg gacaatttta 17220

agactgatca agatcttgct gttcaaatag gtgttaatgg acccattaaa tatgagcatg 17280

ttatctcgtt tatgggtttc cgttttgata tcaacatacc caaccatcat actctctttt 17340

gcacacgcga ctttgccatg cgcaatgtta gaggttggtt aggctttgac gttgaaggag 17400

cacatgttgt tggctctaac gtcggtacaa atgtcccatt gcaattaggg ttttctaacg 17460

gtgttgattt tgttgtcaga cctgaaggtt gcgttgtaac agagtctggt gactacatta 17520

aacccgtcag agctcgtgct ccaccagggg aacaattcgc acaccttttg cctttactta 17580

aacgcggcca accatgggat gttgtccgca aacgtatagt gcagatgtgt agtgactacc 17640

tggccaacct atcagacata ctaatttttg tgttgtgggc tggtggtttg gagttgacaa 17700

ctatgcgtta ttttgtcaag attggaccaa gtaagagttg tgattgtggt aaggttgcta 17760

cttgttacaa tagtgcgctg catacgtact gttgtttcaa acatgccctt ggttgtgatt 17820

atctgtataa cccatactgt attgatatac agcagtgggg atacaaggga tcacttagcc 17880

ttaaccacca tgagcattgt aatgtacata gaaacgagca tgtggcttct ggtgatgcca 17940

taatgactcg ctgtctggcc atacatgatt gctttgtcaa gaacgttgac tggtccatca 18000

catacccatt tattggtaat gaggctgtta ttaataagag cggccgaatt gtgcaatcac 18060

acactatgcg gtcagttctt aagttataca atccgaaagc catatatgat attggcaatc 18120

ctaagggcat tagatgtgcc gtaacggatg ctaagtggtt ttgctttgac aagaatccta 18180

ctaattctaa tgtcaagaca ttggagtatg actatataac acatggccaa tttgatgggt 18240

tgtgcttgtt ttggaattgc aatgtagaca tgtatccaga attttctgtg gtctgtcgtt 18300

ttgatactcg ctgtaggtca ccactcaact tggagggttg taatggtggt tcactgtatg 18360

ttaataatca tgcattccat acaccggctt ttgacaagcg tgcttttgct aagttgaagc 18420

caatgccatt tttcttttat gatgatactg agtgtgacaa gttacaggac tccataaact 18480

atgttcctct tagggctagt aactgcatta ctaaatgtaa tgttggtggt gctgtctgta 18540

gtaagcattg tgctatgtat catagctatg ttaatgctta caacactttt acgtcggcgg 18600

gctttactat ttgggtgcct acttcgtttg acacctataa tctgtggcag acatttagta 18660

acaatttgca aggtcttgag aacattgctt tcaatgtcgt aaagaaagga tcttttgttg 18720

gtgccgaagg tgaacttcct gtagctgtgg ttaatgacaa agtgctcgtt agagatggta 18780

ctgttgatac tcttgttttt acaaacaaga catcactacc cactaacgta gcttttgagt 18840

tgtatgccaa gcgtaaggta ggactcaccc cacccattac gatcctacgt aacttgggtg 18900

tagtttgtac atctaagtgt gtcatttggg actatgaagc cgaacgtcca cttactactt 18960

ttacaaagga tgtttgtaaa tataccgact ttgagggtga cgtctgtaca ctctttgata 19020

acagcattgt tggttcatta gagcgattct ccatgaccca aaatgctgtg cttatgtcac 19080

ttacagctgt taaaaagctt actggcataa agttaactta tggttatctt aatggtgtcc 19140

cagttaacac acatgaagat aaacctttta cttggtatat ttacactagg aagaacggca 19200

agttcgagga ccatcctgat ggctatttta cccaaggtag aacaaccgct gattttagcc 19260

ctcgtagcga catggaaaag gacttcctaa gtatggatat gggtctgttt attaacaagt 19320

acggacttga agattacggc tttgagcacg ttgtgtatgg tgatgtttca aaaaccaccc 19380

ttggtggttt gcatctacta atttcgcagg tgcgtctggc ctgtatgggt gtgctcaaaa 19440

tagacgagtt tgtgtctagt aatgatagca cgttaaagtc ttgtactgtt acatatgctg 19500

ataaccctag tagtaagatg gtttgtacgt atatggatct cctgcttgac gattttgtca 19560

gcattcttaa atctttggat ttgggcgttg tatctaaagt tcatgaagtt atggtcgatt 19620

gtaaaatgtg gaggtggatg ttgtggtgta aggatcataa actccagaca ttttatccgc 19680

aacttcaggc cagtgaatgg aagtgtggtt attccatgcc ttctatttac aagatacaac 19740

gtatgtgttt agaaccttgc aatctctaca actatggtgc tggtattaag ttacctgatg 19800

gcattatgtt taacgtagtt aaatacacac agctttgtca atatctcaat agcaccacaa 19860

tgtgtgtacc ccatcacatg cgtgtgctac atcttggtgc tggctccgac aagggtgttg 19920

cacctggcac ggctgtctta cgacgttggt tgccactgga tgccattata gttgacaatg 19980

atagtgtgga ttacgttagc gatgctgatt atagtgttac aggagattgc tctaccttat 20040

acctgtcaga taagtttgat ttagttatat ctgatatgta tgatggtaag attaaaagtt 20100

gtgatgggga gaacgtgtct aaagaaggct tctttcccta tattaatggt gtcatcaccg 20160

aaaagttggc acttggtggt actgtagcta ttaaggtgac ggagtttagt tggaataaga 20220

agttgtatga actcattcag aggtttgagt attggacaat gttctgtacc agtgttaaca 20280

cgtcatcgtc agaggcattc ttaattggtg ttcactattt aggtgatttt gcaagtggcg 20340

ctgtgattga cggcaacact atgcatgcca attatatctt ctggcgtaat tccacaatta 20400

tgactatgtc ttacaatagt gtacttgatt taagcaagtt caattgtaag cataaggcta 20460

cagttgtcat taatttaaaa gattcatcca ttagtgatgt tgtgttaggt ttgttgaaga 20520

atggtaagtt gctagtgcgt aataatgacg ccatttgtgg tttttctaat catttggtca 20580

acgtaaacaa atgaagtctt taacctactt ctggttgttc ttaccagtac tttcaacact 20640

tagcctacca caagatgtca ccaggtgctc agctaacact aattttaggc ggttcttttc 20700

aaaatttaat gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga 20760

aaaccagggt gtcaattcaa cttggtactg tgctggccaa catccaactg ctagtggcgt 20820

tcatggtatc tttgttagcc atattagagg tggtcatggc tttgagattg gcatttcgca 20880

agagcctttt gaccctagtg gttaccagct ttatttacat aaggctacta acggtaacac 20940

taatgctact gcgcgactgc gcatttgcca gtttcctagc attaataatg atgttacaat 21000

aggtcgtaat tgcctattta acaaagccat cccagctcat atgagtgaac atagtgttgt 21060

cggcataaca tgggataatg atcgtgtcac tgtcttttct gacaagatct attattttta 21120

ttttaaaaat gattggtccc gtgttgcgac aaagtgttac aacagtggag gttgtgctat 21180

gcaatatgtt tacgaaccca cctattacat gcttaatgtt actagtgctg gtgaggatgg 21240

tatttcttat caaccctgta cagctaattg cattggttat gctgccaatg tatttgctac 21300

tgagcccaat ggccacatac cagaaggttt tagttttaat aattggtttc ttttgtccaa 21360

tgattccact ttggtgcatg gtaaggtggt ttccaaccaa ccattgttgg tcaattgtct 21420

tttggccatt cctaagattt atggactagg ccaatttttc tcctttaatc aaacgatcga 21480

tggtgtttgt aatggagctg ctgtgcagcg tgcaccagag gctctgaggt ttaatattaa 21540

tgacacctct gtcattcttg ctgaaggctc aattgtactt catactgctt taggaacaaa 21600

tttttctttt gtttgcagta attcctcaaa tcctcactta gccaccttcg ccatacctct 21660

gggtgctacc caagtacctt attattgttt tcttaaagtg gatacttaca actccactgt 21720

ttataaattt ttggctgttt tacctcctac cgtcagggaa attgtcatca ccaagtatgg 21780

tgatgtttat gtcaatgggt ttggatactt gcatctcggt ttgttggatg ctgtcacaat 21840

taatttcact ggtcatggca ctgacgatga tgtttctggt ttttggacca tagcatcgac 21900

taattttgtt gatgcactca tcgaagttca aggaaccgcc attcagcgta ttctttattg 21960

tgatgatcct gttagccaac tcaagtgttc tcaggttgct tttgaccttg acgatggttt 22020

ttaccctatt tcttctagaa accttctgag tcatgaacag ccaatttctt ttgttactct 22080

gccatcattt aatgatcatt cttttgttaa cattactgta tctgcttcct ttggtggtca 22140

tagtggtgcc aaccttattg catctgacac tactatcaat gggtttagtt ctttctgtgt 22200

tgacactaga caatttacca tttcactgtt ttataacgtt acaaacagtt atggttatgt 22260

gtctaaatca caggacagta attgcccttt caccttgcaa tctgttaatg attacctgtc 22320

ttttagcaaa ttttgtgttt ccaccagcct tttggctagt gcctgtacca tagatctttt 22380

tggttaccct gagtttggta gtggtgttaa gtttacgtcc ctttattttc aattcacaaa 22440

gggtgagttg attactggca cgactaaacc acttgaaggt gtcacggacg tttcttttat 22500

gactctggat gtgtgtacca agtatactat ctatggcttt aaaggtgagg gtatcattac 22560

ccttacaaat tctagctttt tggcaggtgt ttattacaca tctgattctg gacagttgtt 22620

agcctttaag aatgtcacta gtggtgctgt ttattctgtt acgccatgtt ctttttcaga 22680

gcaggctgca tatgttgatg atgatatagt gggtgttatt tctagtttgt ctagctccac 22740

ttttaacagt actagggagt tgcctggttt cttctaccat tctaatgatg gctctaattg 22800

tacagagcct gtgttggtgt atagtaacat aggtgtttgt aaatctggca gtattggcta 22860

cgtcccatct cagtctggcc aagtcaagat tgcacccacg gttactggga atattagtat 22920

tcccaccaac tttagtatga gtattaggac agaatattta cagctttaca acacgcctgt 22980

tagtgttgat tgtgccacat atgtttgtaa tggtaactct cgttgtaaac aattactcac 23040

ccagtacact gcagcatgta agaccataga gtcagcatta caactcagcg ctaggcttga 23100

gtctgttgaa gttaactcta tgcttactat ttctgaagag gctctacagt tagctaccat 23160

tagttcgttt aatggtgatg gatataattt tactaatgtg ctgggtgttt ctgtgtatga 23220

tcctgcaagt ggcagggtgg tacaaaaaag gtcttttatt gaagacctgc tttttaataa 23280

agtggttact aatggccttg gtactgttga tgaagactat aagcgctgtt ctaatggtcg 23340

ctctgtggca gatctagtct gtgcacagta ttactctggt gtcatggtac tacctggtgt 23400

tgttgacgct gagaagcttc acatgtatag tgcgtctctc atcggtggta tggtgctagg 23460

aggttttact tctgcagcgg cattgccttt tagctatgct gttcaagcta gactcaatta 23520

tcttgctcta cagacggatg ttctacagcg gaaccagcaa ttgcttgctg agtcttttaa 23580

ctctgctatt ggtaatataa cttcagcctt tgagagtgtt aaagaggcta ttagtcaaac 23640

ttccaagggt ttgaacactg tggctcatgc gcttactaag gttcaagagg ttgttaactc 23700

gcagggtgca gctttgactc aacttaccgt acagctgcaa cacaacttcc aagccatttc 23760

tagttctatt gatgacattt actctcgact ggacattctt tcagccgatg ttcaggttga 23820

ccgtctcatc accggcagat tatcagcact taatgctttt gttgctcaaa ccctcactaa 23880

gtatactgag gttcaggcta gcaggaagtt agcacagcaa aaggttaatg agtgcgttaa 23940

atcgcaatct cagcgttatg gtttttgtgg tggtgatggc gagcacattt tctctctggt 24000

acaggcagca cctcagggcc tgctgttttt acatacagta cttgtaccga gtgattttgt 24060

agatgttatt gccatcgctg gcttatgcgt taacgatgaa attgccttga ctctacgtga 24120

gcctggctta gtcttgttta cgcatgaact tcaaaatcat actgcgacgg aatattttgt 24180

ttcatcgcga cgtatgtttg aacctagaaa acctaccgtt agtgattttg ttcaaattga 24240

gagttgtgtg gtcacctatg tcaatttgac tagagaccaa ctaccagatg taatcccaga 24300

ttacatcgat gttaacaaaa cacttgatga gattttagct tctctgccca atagaactgg 24360

tccaagtctt cctttagatg tttttaatgc cacttatctt aatctcactg gtgaaattgc 24420

agatttagag cagcgttcag agtctctccg taatactaca gaggagctcc aaagtcttat 24480

atataatatc aacaacacac tagttgacct tgagtggctc aaccgagttg agacatatat 24540

caagtggccg tggtgggttt ggttgattat tttcattgtt ctcatctttg ttgtgtcatt 24600

actagtgttc tgctgcattt ccacgggttg ttgtggatgc tgcggctgct gctgtgcttg 24660

tttctcaggt tgttgtaggg gtcctagact tcaaccttac gaagtttttg aaaaggtcca 24720

cgtgcagtga tgtttcttgg actttttcaa tacacgattg acacagttgt caaagatgtc 24780

tcaaagtctg ctaacttgtc tttggatgct gtccaagagt tggagctcaa tgtagttcca 24840

attagacaag cttcaaatgt gacgggtttt cttttcacca gtgtttttat ctacttcttt 24900

gcactgttta aagcgtcttc tttgaggcgc aattatatta tgttggcagc gcgttttgct 24960

gtcattgttc tttattgccc acttttatat tattgtggtg catttttaga tgcaactatt 25020

atttgttgca cacttattgg caggctttgt ttagtctgct tttactcctg gcgctataaa 25080

aatgcgctct ttattatttt taatactacg acactttctt tcctcaatgg taaagcagct 25140

tattatgacg gcaaatccat tgtgatttta gaaggtggtg accattacat cacttttggc 25200

aactcttttg ttgcttttgt tagtagcatc gacttgtatc tagctatacg tgggcggcaa 25260

gaagctgacc tacagctgtt gcgaactgtt gagcttcttg atggcaagaa gctttatgtc 25320

ttttcgcaac atcaaattgt tggcattact aatgctgcat ttgactcaat tcaactagac 25380

gagtatgcta caattagtga atgataatgg tctagtagtt aatgttatac tttggctttt 25440

cgtactcttt ttcctgctta ttataagcat tactttcgtc caattggtta atctgtgctt 25500

cacttgtcac cggttgtgta atagcgcagt ttacacacct atagggcgtt tgtatagagt 25560

ttataagtct tacatgcaaa tagaccccct ccctagtact gttattgacg tataaacgaa 25620

atatgtctaa cggttctatt cccgttgatg aggtgattca acaccttaga aactggaatt 25680

tcacatggaa tatcatactg acgatactac ttgtagtgct tcagtatggc cattacaagt 25740

actctgcgtt cttgtatggt gtcaagatgg ctattctatg gatactttgg cctcttgtgt 25800

tagcactgtc actttttgat gcatgggcta gctttcaggt caattgggtc ttttttgctt 25860

tcagcatcct tatggcttgc atcactctta tgctgtggat aatgtacttt gtcaatagca 25920

ttcggttgtg gcgcaggaca cattcttggt ggtctttcaa tcctgaaaca gacgcgcttc 25980

tcactacttc tgtgatgggc cgacaggtct gcattccagt gcttggagca ccaactggtg 26040

taacgctaac actccttagt ggtacattgc ttgtagaggg ctataaggtt gctactggcg 26100

tacaggtaag tcaattacct aatttcgtca cagtcgccaa ggccactaca acaattgtct 26160

acggacgtgt tggtcgttca gtcaatgctt catctggcac tggttgggct ttctatgtcc 26220

ggtccaaaca cggcgactac tcagctgtga gtaatccgag ttcggttctc acagatagtg 26280

agaaagtgct tcatttagtc taaacagaaa ctttatggct tctgtcagtt ttcaggatcg 26340

tggccgcaaa cgggtgccat tatccctcta tgcccctctt agggttacta atgacaaacc 26400

cctttctaag gtacttgcaa ataatgctgt acccactaat aaaggaaata aggaccagca 26460

aattggatac tggaatgagc aaattcgctg gcgcatgcgc cgtggtgagc gaattgaaca 26520

accttccaat tggcatttct actacctcgg aacaggacct cacgccgacc tccgctacag 26580

gactcgtact gagggtgttt tctgggttgc taaagaaggc gcaaagactg aacccactaa 26640

cctgggtgtc agaaaggcgt ctgaaaagcc aattattcca aatttctctc aacagcttcc 26700

cagcgtagtt gagattgttg aacctaacac acctcctact tcacgtgcaa attcacgtag 26760

caggagtcgt ggtaatggca acaacaggtc cagatctcca agtaacaaca gaggcaataa 26820

ccagtcccgc ggtaattcac agaatcgtgg aaataaccag ggtcgtggag cttctcagaa 26880

cagaggaggc aataataata acaataacaa gtctcgtaac cagtccaaga acagaaacca 26940

gtcaaatgac cgtggtggtg taacatcacg cgatgatctg gtggctgctg tcaaggatgc 27000

ccttaaatct ttgggtattg gcgaaaaccc tgacaagctt aagcaacagc agaagcccaa 27060

acaggaaagg tctgacagca gcggcaaaaa tacacctaag aagaacaaat ccagagccac 27120

ttcgaaagaa cgtgacctca aagacatccc agagtggagg agaattccca agggcgaaaa 27180

tagcgtagca gcttgcttcg gacccagggg aggcttcaaa aattttggag atgcggaatt 27240

tgtcgaaaaa ggtgttgatg cctcaggcta tgctcagatc gccagtttag caccaaatgt 27300

tgcagcattg ctctttggtg gtaatgtggc tgttcgtgag ctagcggact cttacgagat 27360

tacatataat tataaaatga ctgtgccaaa gtctgatcca aatgtagagc ttcttgtttc 27420

acaggtggat gcatttaaaa ctgggaatgc aaaaccccag agaaagaagg aaaagaagaa 27480

caagcgtgaa accacgcagc agctgaatga agaggccatc tacgatgatg tgggtgtgcc 27540

atctgatgtg actcatgcca atttggaatg ggacacagct gttgatggtg gtgacacggc 27600

cgttgaaatt atcaacgaga tcttcgacac aggaaattaa acaatgtttg actggcttat 27660

cctggctatg tcccagggta gtgccattac actgttatta ctgagtgttt ttctagcgac 27720

ttggctgctg ggctatggct ttgccctcta actagcggtc ttggtcttgc acacaacggt 27780

aagccagtgg taatgtcagt gcaagaagga tattaccata gcactgtcat gaggggaacg 27840

cagtaccttt tcatctaaac ctttgcacga gtaatcaaag atccgcttga cgagcctata 27900

tggaagagcg tgccaggtat ttgactcaag gactgttagt aactgaagac ctgacggtgt 27960

<210> 30

<211> 4140

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV Isolate 1251-125-10 (125-10) Spike-protein cDNA Sequence

<400> 30

atgaagtctt taacctactt ctggttgttc ttaccagtac tttcaacact tagcctacca 60

caagatgtca ccaggtgctc agctaacact aattttaggc ggttcttttc aaaatttaat 120

gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga aaaccagggt 180

gtcaattcaa cttggtactg tgctggccaa catccaactg ctagtggcgt tcatggtatc 240

tttgttagcc atattagagg tggtcatggc tttgagattg gcatttcgca agagcctttt 300

gaccctagtg gttaccagct ttatttacat aaggctacta acggtaacac taatgctact 360

gcgcgactgc gcatttgcca gtttcctagc attaataatg atgttacaat aggtcgtaat 420

tgcctattta acaaagccat cccagctcat atgagtgaac atagtgttgt cggcataaca 480

tgggataatg atcgtgtcac tgtcttttct gacaagatct attattttta ttttaaaaat 540

gattggtccc gtgttgcgac aaagtgttac aacagtggag gttgtgctat gcaatatgtt 600

tacgaaccca cctattacat gcttaatgtt actagtgctg gtgaggatgg tatttcttat 660

caaccctgta cagctaattg cattggttat gctgccaatg tatttgctac tgagcccaat 720

ggccacatac cagaaggttt tagttttaat aattggtttc ttttgtccaa tgattccact 780

ttggtgcatg gtaaggtggt ttccaaccaa ccattgttgg tcaattgtct tttggccatt 840

cctaagattt atggactagg ccaatttttc tcctttaatc aaacgatcga tggtgtttgt 900

aatggagctg ctgtgcagcg tgcaccagag gctctgaggt ttaatattaa tgacacctct 960

gtcattcttg ctgaaggctc aattgtactt catactgctt taggaacaaa tttttctttt 1020

gtttgcagta attcctcaaa tcctcactta gccaccttcg ccatacctct gggtgctacc 1080

caagtacctt attattgttt tcttaaagtg gatacttaca actccactgt ttataaattt 1140

ttggctgttt tacctcctac cgtcagggaa attgtcatca ccaagtatgg tgatgtttat 1200

gtcaatgggt ttggatactt gcatctcggt ttgttggatg ctgtcacaat taatttcact 1260

ggtcatggca ctgacgatga tgtttctggt ttttggacca tagcatcgac taattttgtt 1320

gatgcactca tcgaagttca aggaaccgcc attcagcgta ttctttattg tgatgatcct 1380

gttagccaac tcaagtgttc tcaggttgct tttgaccttg acgatggttt ttaccctatt 1440

tcttctagaa accttctgag tcatgaacag ccaatttctt ttgttactct gccatcattt 1500

aatgatcatt cttttgttaa cattactgta tctgcttcct ttggtggtca tagtggtgcc 1560

aaccttattg catctgacac tactatcaat gggtttagtt ctttctgtgt tgacactaga 1620

caatttacca tttcactgtt ttataacgtt acaaacagtt atggttatgt gtctaaatca 1680

caggacagta attgcccttt caccttgcaa tctgttaatg attacctgtc ttttagcaaa 1740

ttttgtgttt ccaccagcct tttggctagt gcctgtacca tagatctttt tggttaccct 1800

gagtttggta gtggtgttaa gtttacgtcc ctttattttc aattcacaaa gggtgagttg 1860

attactggca cgactaaacc acttgaaggt gtcacggacg tttcttttat gactctggat 1920

gtgtgtacca agtatactat ctatggcttt aaaggtgagg gtatcattac ccttacaaat 1980

tctagctttt tggcaggtgt ttattacaca tctgattctg gacagttgtt agcctttaag 2040

aatgtcacta gtggtgctgt ttattctgtt acgccatgtt ctttttcaga gcaggctgca 2100

tatgttgatg atgatatagt gggtgttatt tctagtttgt ctagctccac ttttaacagt 2160

actagggagt tgcctggttt cttctaccat tctaatgatg gctctaattg tacagagcct 2220

gtgttggtgt atagtaacat aggtgtttgt aaatctggca gtattggcta cgtcccatct 2280

cagtctggcc aagtcaagat tgcacccacg gttactggga atattagtat tcccaccaac 2340

tttagtatga gtattaggac agaatattta cagctttaca acacgcctgt tagtgttgat 2400

tgtgccacat atgtttgtaa tggtaactct cgttgtaaac aattactcac ccagtacact 2460

gcagcatgta agaccataga gtcagcatta caactcagcg ctaggcttga gtctgttgaa 2520

gttaactcta tgcttactat ttctgaagag gctctacagt tagctaccat tagttcgttt 2580

aatggtgatg gatataattt tactaatgtg ctgggtgttt ctgtgtatga tcctgcaagt 2640

ggcagggtgg tacaaaaaag gtcttttatt gaagacctgc tttttaataa agtggttact 2700

aatggccttg gtactgttga tgaagactat aagcgctgtt ctaatggtcg ctctgtggca 2760

gatctagtct gtgcacagta ttactctggt gtcatggtac tacctggtgt tgttgacgct 2820

gagaagcttc acatgtatag tgcgtctctc atcggtggta tggtgctagg aggttttact 2880

tctgcagcgg cattgccttt tagctatgct gttcaagcta gactcaatta tcttgctcta 2940

cagacggatg ttctacagcg gaaccagcaa ttgcttgctg agtcttttaa ctctgctatt 3000

ggtaatataa cttcagcctt tgagagtgtt aaagaggcta ttagtcaaac ttccaagggt 3060

ttgaacactg tggctcatgc gcttactaag gttcaagagg ttgttaactc gcagggtgca 3120

gctttgactc aacttaccgt acagctgcaa cacaacttcc aagccatttc tagttctatt 3180

gatgacattt actctcgact ggacattctt tcagccgatg ttcaggttga ccgtctcatc 3240

accggcagat tatcagcact taatgctttt gttgctcaaa ccctcactaa gtatactgag 3300

gttcaggcta gcaggaagtt agcacagcaa aaggttaatg agtgcgttaa atcgcaatct 3360

cagcgttatg gtttttgtgg tggtgatggc gagcacattt tctctctggt acaggcagca 3420

cctcagggcc tgctgttttt acatacagta cttgtaccga gtgattttgt agatgttatt 3480

gccatcgctg gcttatgcgt taacgatgaa attgccttga ctctacgtga gcctggctta 3540

gtcttgttta cgcatgaact tcaaaatcat actgcgacgg aatattttgt ttcatcgcga 3600

cgtatgtttg aacctagaaa acctaccgtt agtgattttg ttcaaattga gagttgtgtg 3660

gtcacctatg tcaatttgac tagagaccaa ctaccagatg taatcccaga ttacatcgat 3720

gttaacaaaa cacttgatga gattttagct tctctgccca atagaactgg tccaagtctt 3780

cctttagatg tttttaatgc cacttatctt aatctcactg gtgaaattgc agatttagag 3840

cagcgttcag agtctctccg taatactaca gaggagctcc aaagtcttat atataatatc 3900

aacaacacac tagttgacct tgagtggctc aaccgagttg agacatatat caagtggccg 3960

tggtgggttt ggttgattat tttcattgtt ctcatctttg ttgtgtcatt actagtgttc 4020

tgctgcattt ccacgggttg ttgtggatgc tgcggctgct gctgtgcttg tttctcaggt 4080

tgttgtaggg gtcctagact tcaaccttac gaagtttttg aaaaggtcca cgtgcagtga 4140

<210> 31

<211> 1379

<212> PRT

<213> porcine epidemic diarrhea virus

<400> 31

Met Lys Ser Leu Thr Tyr Phe Trp Leu Phe Leu Pro Val Leu Ser Thr

1 5 10 15

Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe

20 25 30

Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val

35 40 45

Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr

50 55 60

Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly Ile

65 70 75 80

Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser

85 90 95

Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala

100 105 110

Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe

115 120 125

Pro Ser Ile Asn Asn Asp Val Thr Ile Gly Arg Asn Cys Leu Phe Asn

130 135 140

Lys Ala Ile Pro Ala His Met Ser Glu His Ser Val Val Gly Ile Thr

145 150 155 160

Trp Asp Asn Asp Arg Val Thr Val Phe Ser Asp Lys Ile Tyr Tyr Phe

165 170 175

Tyr Phe Lys Asn Asp Trp Ser Arg Val Ala Thr Lys Cys Tyr Asn Ser

180 185 190

Gly Gly Cys Ala Met Gln Tyr Val Tyr Glu Pro Thr Tyr Tyr Met Leu

195 200 205

Asn Val Thr Ser Ala Gly Glu Asp Gly Ile Ser Tyr Gln Pro Cys Thr

210 215 220

Ala Asn Cys Ile Gly Tyr Ala Ala Asn Val Phe Ala Thr Glu Pro Asn

225 230 235 240

Gly His Ile Pro Glu Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu Ser

245 250 255

Asn Asp Ser Thr Leu Val His Gly Lys Val Val Ser Asn Gln Pro Leu

260 265 270

Leu Val Asn Cys Leu Leu Ala Ile Pro Lys Ile Tyr Gly Leu Gly Gln

275 280 285

Phe Phe Ser Phe Asn Gln Thr Ile Asp Gly Val Cys Asn Gly Ala Ala

290 295 300

Val Gln Arg Ala Pro Glu Ala Leu Arg Phe Asn Ile Asn Asp Thr Ser

305 310 315 320

Val Ile Leu Ala Glu Gly Ser Ile Val Leu His Thr Ala Leu Gly Thr

325 330 335

Asn Phe Ser Phe Val Cys Ser Asn Ser Ser Asn Pro His Leu Ala Thr

340 345 350

Phe Ala Ile Pro Leu Gly Ala Thr Gln Val Pro Tyr Tyr Cys Phe Leu

355 360 365

Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr Lys Phe Leu Ala Val Leu

370 375 380

Pro Pro Thr Val Arg Glu Ile Val Ile Thr Lys Tyr Gly Asp Val Tyr

385 390 395 400

Val Asn Gly Phe Gly Tyr Leu His Leu Gly Leu Leu Asp Ala Val Thr

405 410 415

Ile Asn Phe Thr Gly His Gly Thr Asp Asp Asp Val Ser Gly Phe Trp

420 425 430

Thr Ile Ala Ser Thr Asn Phe Val Asp Ala Leu Ile Glu Val Gln Gly

435 440 445

Thr Ala Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val Ser Gln Leu

450 455 460

Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe Tyr Pro Ile

465 470 475 480

Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser Phe Val Thr

485 490 495

Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr Val Ser Ala

500 505 510

Ser Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala Ser Asp Thr Thr

515 520 525

Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg Gln Phe Thr Ile

530 535 540

Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr Val Ser Lys Ser

545 550 555 560

Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn Asp Tyr Leu

565 570 575

Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala Ser Ala Cys

580 585 590

Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser Gly Val Lys Phe

595 600 605

Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile Thr Gly Thr

610 615 620

Thr Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met Thr Leu Asp

625 630 635 640

Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly Glu Gly Ile Ile

645 650 655

Thr Leu Thr Asn Ser Ser Phe Leu Ala Gly Val Tyr Tyr Thr Ser Asp

660 665 670

Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser Gly Ala Val Tyr

675 680 685

Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala Tyr Val Asp Asp

690 695 700

Asp Ile Val Gly Val Ile Ser Ser Leu Ser Ser Ser Thr Phe Asn Ser

705 710 715 720

Thr Arg Glu Leu Pro Gly Phe Phe Tyr His Ser Asn Asp Gly Ser Asn

725 730 735

Cys Thr Glu Pro Val Leu Val Tyr Ser Asn Ile Gly Val Cys Lys Ser

740 745 750

Gly Ser Ile Gly Tyr Val Pro Ser Gln Ser Gly Gln Val Lys Ile Ala

755 760 765

Pro Thr Val Thr Gly Asn Ile Ser Ile Pro Thr Asn Phe Ser Met Ser

770 775 780

Ile Arg Thr Glu Tyr Leu Gln Leu Tyr Asn Thr Pro Val Ser Val Asp

785 790 795 800

Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser Arg Cys Lys Gln Leu Leu

805 810 815

Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Ala Leu Gln Leu

820 825 830

Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu Thr Ile Ser

835 840 845

Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn Gly Asp Gly

850 855 860

Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp Pro Ala Ser

865 870 875 880

Gly Arg Val Val Gln Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn

885 890 895

Lys Val Val Thr Asn Gly Leu Gly Thr Val Asp Glu Asp Tyr Lys Arg

900 905 910

Cys Ser Asn Gly Arg Ser Val Ala Asp Leu Val Cys Ala Gln Tyr Tyr

915 920 925

Ser Gly Val Met Val Leu Pro Gly Val Val Asp Ala Glu Lys Leu His

930 935 940

Met Tyr Ser Ala Ser Leu Ile Gly Gly Met Val Leu Gly Gly Phe Thr

945 950 955 960

Ser Ala Ala Ala Leu Pro Phe Ser Tyr Ala Val Gln Ala Arg Leu Asn

965 970 975

Tyr Leu Ala Leu Gln Thr Asp Val Leu Gln Arg Asn Gln Gln Leu Leu

980 985 990

Ala Glu Ser Phe Asn Ser Ala Ile Gly Asn Ile Thr Ser Ala Phe Glu

995 1000 1005

Ser Val Lys Glu Ala Ile Ser Gln Thr Ser Lys Gly Leu Asn Thr

1010 1015 1020

Val Ala His Ala Leu Thr Lys Val Gln Glu Val Val Asn Ser Gln

1025 1030 1035

Gly Ala Ala Leu Thr Gln Leu Thr Val Gln Leu Gln His Asn Phe

1040 1045 1050

Gln Ala Ile Ser Ser Ser Ile Asp Asp Ile Tyr Ser Arg Leu Asp

1055 1060 1065

Ile Leu Ser Ala Asp Val Gln Val Asp Arg Leu Ile Thr Gly Arg

1070 1075 1080

Leu Ser Ala Leu Asn Ala Phe Val Ala Gln Thr Leu Thr Lys Tyr

1085 1090 1095

Thr Glu Val Gln Ala Ser Arg Lys Leu Ala Gln Gln Lys Val Asn

1100 1105 1110

Glu Cys Val Lys Ser Gln Ser Gln Arg Tyr Gly Phe Cys Gly Gly

1115 1120 1125

Asp Gly Glu His Ile Phe Ser Leu Val Gln Ala Ala Pro Gln Gly

1130 1135 1140

Leu Leu Phe Leu His Thr Val Leu Val Pro Ser Asp Phe Val Asp

1145 1150 1155

Val Ile Ala Ile Ala Gly Leu Cys Val Asn Asp Glu Ile Ala Leu

1160 1165 1170

Thr Leu Arg Glu Pro Gly Leu Val Leu Phe Thr His Glu Leu Gln

1175 1180 1185

Asn His Thr Ala Thr Glu Tyr Phe Val Ser Ser Arg Arg Met Phe

1190 1195 1200

Glu Pro Arg Lys Pro Thr Val Ser Asp Phe Val Gln Ile Glu Ser

1205 1210 1215

Cys Val Val Thr Tyr Val Asn Leu Thr Arg Asp Gln Leu Pro Asp

1220 1225 1230

Val Ile Pro Asp Tyr Ile Asp Val Asn Lys Thr Leu Asp Glu Ile

1235 1240 1245

Leu Ala Ser Leu Pro Asn Arg Thr Gly Pro Ser Leu Pro Leu Asp

1250 1255 1260

Val Phe Asn Ala Thr Tyr Leu Asn Leu Thr Gly Glu Ile Ala Asp

1265 1270 1275

Leu Glu Gln Arg Ser Glu Ser Leu Arg Asn Thr Thr Glu Glu Leu

1280 1285 1290

Gln Ser Leu Ile Tyr Asn Ile Asn Asn Thr Leu Val Asp Leu Glu

1295 1300 1305

Trp Leu Asn Arg Val Glu Thr Tyr Ile Lys Trp Pro Trp Trp Val

1310 1315 1320

Trp Leu Ile Ile Phe Ile Val Leu Ile Phe Val Val Ser Leu Leu

1325 1330 1335

Val Phe Cys Cys Ile Ser Thr Gly Cys Cys Gly Cys Cys Gly Cys

1340 1345 1350

Cys Cys Ala Cys Phe Ser Gly Cys Cys Arg Gly Pro Arg Leu Gln

1355 1360 1365

Pro Tyr Glu Val Phe Glu Lys Val His Val Gln

1370 1375

<210> 32

<211> 27960

<212> RNA

<213> porcine epidemic diarrhea virus

<400> 32

agacucuugu cuacucaauu caacuaaacg aaauuuuguc cuuccggccg cauguccaug 60

cugcuggaag cugacgugga auuucauuag guuugcuuaa guagccaucg caagugcugu 120

gcuguccucu aguuccuggu uggcguuccg ucgccuucua cauacuagac aaacagccuu 180

ccuccgguuc cgucuggggg uuguguggau aacuaguucc gucuaguuug aaaccaguaa 240

cugucggcua uggcuagcaa ccauguuaca uuggcuuuug ccaaugaugc agaaauuuca 300

gcuuuuggcu uuugcacugc uagugaagcc gucucauacu auucugaggc cgccgcuagu 360

ggauuuaugc aaugccguuu cguguccuuc gaucucgcug acacuguuga gggauugcuu 420

cccgaagacu augucauggu gguggucggc acuaccaagc uuagugcgua uguggacacu 480

uuugguagcc gccccaaaaa cauuuguggu uggcuguuau uuucuaacug uaauuacuuc 540

cucgaagagu uagagcuuac uuuuggucgu cgugguggua acaucgugcc aguugaccaa 600

uacaugugug gcgcugacgg uaaaccuguu cuucaggaau ccgaauggga guauacagau 660

uucuuugcug acuccgaaga cggucaacuc aacauugcug guaucacuua ugugaaggcc 720

uggauuguag agcgaucgga ugucucuuau gcgagucaga auuuaacauc uauuaagucu 780

auuacuuacu guucaaccua ugagcauacu uuuccugaug guacugccau gaagguugca 840

cguacuccaa agauuaagaa gacuguuguc uugucugagc cacuugcuac uaucuacagg 900

gaaauugguu cuccuuuugu ggauaauggg agcgaugcuc guucuaucau uaagagacca 960

guguuccucc acgcuuuugu uaaguguaag ugugguaguu aucauuggac uguuggugau 1020

uggacuuccu augucuccac uugcuguggc uuuaagugua agccaguccu uguggcuuca 1080

ugcucugcua cgccugguuc uguugugguu acgcgcgcug gugcuggcac ugguguuaag 1140

uauuacaaca acauguuccu gcgccaugug gcagacauug auggguuggc auucuggcga 1200

auucucaagg ugcaguccaa agacgaccuc gcuugcucug guaaauuccu ugaacaccau 1260

gaggaagguu ucacagaucc uugcuacuuu uugaaugacu cgagcauugc uacuaagcuc 1320

aaguuugaca uccuuagugg caaguuuucu gaugaaguca aacaagcuau cuuugcuggu 1380

cauguuguug uuggcagcgc gcucguugac auuguugacg augcacuggg acagccuugg 1440

uuuauacgua agcuugguga ccuugcaagu gcagcuuggg agcagcuuaa ggcugucguu 1500

agaggccuua accuccuguc ugaugagguc gugcucuuug gcaaaagacu uagcugugcc 1560

acucuuagua ucguuaacgg uguuuuugag uucaucgccg aagugccuga gaaguuggcu 1620

gcggcuguua caguuuuugu caacuucuug aaugagcuuu uugagucugc cugugacugc 1680

uuaaaggucg gagguaaaac cuuuaacaag guuggcucuu auguucuuuu ugacaacgca 1740

uugguuaagc uugucaaggc aaaaguucgc ggcccacgac aggcaggugu uugugaaguu 1800

cguuacacaa gccuuguuau ugggaguacu accaaggugg uuuccaagcg cguugaaaau 1860

gccaauguga aucucgucgu cguugacgag gaugugaccc ucaacaccac uggucguaca 1920

guuguuguug acggacuugc auucuucgag agugacgggu uuuacagaca ucuugcugau 1980

gcugacguug ucauugaaca uccuguuuau aagucugcuu gugagcucaa gccaguuuuu 2040

gagugugacc caauaccuga uuuuccuaug ccuguggccg cuaguguugc agagcuuugu 2100

gugcaaacug aucuguugcu uaaaaauuac aacacuccuu auaaaacuua cagcugcguu 2160

gugagaggug auaaguguug uaucacuugc accuuacauu ucacagcacc aaguuauaug 2220

gaggcugcug cuaauuuugu agaccucugu accaagaaca uugguacugc ugguuuucau 2280

gaguuuuaca uuacggccca ugaacaacag gaucugcaag gguucguaac cacuuguugc 2340

acgaugucag guuuugagug uuuuaugccu auaaucccac aguguccagc agugcuugaa 2400

gagauugaug gugguagcau cuggcggucu uuuaucacug gucuuaauac aaugugggau 2460

uuuugcaagc aucuuaaagu cagcuuugga cuagauggca uuguugucac uguagcacgc 2520

aaauuuaaac gacuuggugc ucucuuggca gaaauguaua acacuuaccu uucaacugug 2580

guggaaaacu ugguacuggc cgguguuagc uucaaguauu augccaccag ugucccaaaa 2640

auuguuuugg gcuguuguuu ucacaguguu aaaaguguuc uugcaagugc cuuccagauu 2700

ccuguccagg caggcguuga gaaguuuaaa gucuuccuua acuguguuca cccuguugua 2760

ccacguguca uugaaacuuc uuuuguggaa uuagaagaga cgacauuuaa accaccagca 2820

cucaauggua guauugcuau uguugauggc uuugcuuucu auuaugaugg aacacuauac 2880

uaucccaccg augguaauag cguuguuccu aucugcuuua agaagaaagg ugguggugau 2940

gucaaauucu cugaugaagu cucuguuaaa accauugacc caguuuauaa ggucucccuu 3000

gaauuugagu ucgagucuga gacuauuaug gcugugcuua auaaggcugu ugguaauugu 3060

aucaagguua caggugguug ggacgauguu guugaguaua ucaauguugc cauugagguu 3120

cuuaaagauc acaucgaugu gccuaaguac uacaucuaug augaggaagg uggcaccgau 3180

ccuaaucugc ccguaauggu uucucagugg ccguugaaug augacacgau cucacaggau 3240

cugcuugaug uugaaguugu uacugaugcg ccaguugauu ucgaggguga ugaaguagac 3300

uccucugacc cugauaaggu ggcagacgug gcuaacucug agccugagga ugacggucuu 3360

aauguagcuc cugaaacaaa uguagagucu gaaguugagg aaguugccgc aaccuugucc 3420

uuuauuaaag auacaccuuc cacaguuacu aaggauccuu uugcuuuuga cuuugcaagc 3480

uauggaggac uuaagguuuu aagacaaucu cauaacaacu gcuggguuac uucuaccuug 3540

gugcagcuac aauugcuugg caucguugau gacccugcaa uggagcuuuu uagugcuggu 3600

agaguugguc caaugguucg caaaugcuau gagucacaaa aggcuaucuu gggaucuuug 3660

ggugaugugu cggcuugccu agagucucug acuaaggacc uacacacacu uaagauuacc 3720

uguucuguag ucugugguug ugguacuggu gaacguaucu augaugguug ugcuuuucgu 3780

augacgccaa cuuuggaacc guucccauau ggugcuugug cucagugugc ucaaguuuug 3840

augcacacuu uuaaaaguau uguuggcacc ggcaucuuuu gucgagauac uacugcucuc 3900

uccuuggauu cuuugguugu aaaaccucuu ugugcggcug cuuuuauagg caaggauagu 3960

ggucauuaug ucacuaacuu uuaugaugcu gcuauggcua uugaugguua uggucgucau 4020

cagauaaagu augacacacu gaacacuauu uguguuaaag acguuaauug gacagcaccu 4080

uuugucccag acguugagcc uguauuggag ccuguuguca aaccuuucua uucuuauaag 4140

aauguugauu uuuaccaagg agauuuuagu gaccuuguua aacuuccaug ugauuuuguu 4200

guuaaugcug caaaugagaa uuugucucac gguggcggca uagcaaaggc cauugauguu 4260

uauaccaagg gcauguugca gaagugcucg aaugauuaca uuaaagcaca cggucccauu 4320

aaaguuggac guggugucau guuggaggca uuaggucuua aggucuuuaa uguuguuggu 4380

ccacguaagg guaagcaugc accugagcuu cuuguuaagg cuuauaaguc cguuuuugcu 4440

aauucaggug uugcucuuac accuuugauu aguguuggaa uuuuuagugu uccuuuggaa 4500

gaaucuuuau cugcuuuucu ugcauguguu ggugaucgcc acuguaagug cuuuuguuau 4560

agugacaaag agcgcgaggc gaucauuaau uacauggaug gcuugguaga ugcuauuuuc 4620

aaagaugcac uuguugauac uacuccuguc caggaagaug uucaacaagu uucacaaaaa 4680

ccaguuuugc cuaauuuuga accuuucagg auugaaggug cucaugcuuu cuaugagugc 4740

aacccugaag guuugauguc auuaggugcu gacaagcugg uguuguuuac aaauuccaau 4800

uuggauuuuu guagcguugg uaagugucuu aacaauguga cuggcggugc auugcuugaa 4860

gccauaaaug uauuuaaaaa gaguaacaaa acagugccug cuggcaacug uguuacuuuu 4920

gagugugcag auaugauuuc uauuacuaug guaguauugc caucugacgg ugaugcuaau 4980

uaugacaaaa auuaugcacg cgccgucguc aagguaucua agcuuaaagg caaguuauug 5040

cuugcuguug gugaugccau guuguauucc aaguuguccc accucagcgu guuagguuuc 5100

guauccacac cugaugaugu ggagcguuuc uacgcaaaua agaguguggu uauuaaaguu 5160

acugaggaua cacguagugu uaagacuguu aaaguagaau ccacuguuac uuauggacaa 5220

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cuguugauac ucuuguuuuu acaaacaaga caucacuacc cacuaacgua gcuuuugagu 18840

uguaugccaa gcguaaggua ggacucaccc cacccauuac gauccuacgu aacuugggug 18900

uaguuuguac aucuaagugu gucauuuggg acuaugaagc cgaacgucca cuuacuacuu 18960

uuacaaagga uguuuguaaa uauaccgacu uugaggguga cgucuguaca cucuuugaua 19020

acagcauugu ugguucauua gagcgauucu ccaugaccca aaaugcugug cuuaugucac 19080

uuacagcugu uaaaaagcuu acuggcauaa aguuaacuua ugguuaucuu aauggugucc 19140

caguuaacac acaugaagau aaaccuuuua cuugguauau uuacacuagg aagaacggca 19200

aguucgagga ccauccugau ggcuauuuua cccaagguag aacaaccgcu gauuuuagcc 19260

cucguagcga cauggaaaag gacuuccuaa guauggauau gggucuguuu auuaacaagu 19320

acggacuuga agauuacggc uuugagcacg uuguguaugg ugauguuuca aaaaccaccc 19380

uuggugguuu gcaucuacua auuucgcagg ugcgucuggc cuguaugggu gugcucaaaa 19440

uagacgaguu ugugucuagu aaugauagca cguuaaaguc uuguacuguu acauaugcug 19500

auaacccuag uaguaagaug guuuguacgu auauggaucu ccugcuugac gauuuuguca 19560

gcauucuuaa aucuuuggau uugggcguug uaucuaaagu ucaugaaguu auggucgauu 19620

guaaaaugug gagguggaug uuguggugua aggaucauaa acuccagaca uuuuauccgc 19680

aacuucaggc cagugaaugg aagugugguu auuccaugcc uucuauuuac aagauacaac 19740

guauguguuu agaaccuugc aaucucuaca acuauggugc ugguauuaag uuaccugaug 19800

gcauuauguu uaacguaguu aaauacacac agcuuuguca auaucucaau agcaccacaa 19860

uguguguacc ccaucacaug cgugugcuac aucuuggugc uggcuccgac aaggguguug 19920

caccuggcac ggcugucuua cgacguuggu ugccacugga ugccauuaua guugacaaug 19980

auagugugga uuacguuagc gaugcugauu auaguguuac aggagauugc ucuaccuuau 20040

accugucaga uaaguuugau uuaguuauau cugauaugua ugaugguaag auuaaaaguu 20100

gugaugggga gaacgugucu aaagaaggcu ucuuucccua uauuaauggu gucaucaccg 20160

aaaaguuggc acuugguggu acuguagcua uuaaggugac ggaguuuagu uggaauaaga 20220

aguuguauga acucauucag agguuugagu auuggacaau guucuguacc aguguuaaca 20280

cgucaucguc agaggcauuc uuaauuggug uucacuauuu aggugauuuu gcaaguggcg 20340

cugugauuga cggcaacacu augcaugcca auuauaucuu cuggcguaau uccacaauua 20400

ugacuauguc uuacaauagu guacuugauu uaagcaaguu caauuguaag cauaaggcua 20460

caguugucau uaauuuaaaa gauucaucca uuagugaugu uguguuaggu uuguugaaga 20520

augguaaguu gcuagugcgu aauaaugacg ccauuugugg uuuuucuaau cauuugguca 20580

acguaaacaa augaagucuu uaaccuacuu cugguuguuc uuaccaguac uuucaacacu 20640

uagccuacca caagauguca ccaggugcuc agcuaacacu aauuuuaggc gguucuuuuc 20700

aaaauuuaau guucaggcgc cugcaguugu uguacugggc gguuaucuac cuauugguga 20760

aaaccagggu gucaauucaa cuugguacug ugcuggccaa cauccaacug cuaguggcgu 20820

ucaugguauc uuuguuagcc auauuagagg uggucauggc uuugagauug gcauuucgca 20880

agagccuuuu gacccuagug guuaccagcu uuauuuacau aaggcuacua acgguaacac 20940

uaaugcuacu gcgcgacugc gcauuugcca guuuccuagc auuaauaaug auguuacaau 21000

aggucguaau ugccuauuua acaaagccau cccagcucau augagugaac auaguguugu 21060

cggcauaaca ugggauaaug aucgugucac ugucuuuucu gacaagaucu auuauuuuua 21120

uuuuaaaaau gauugguccc guguugcgac aaaguguuac aacaguggag guugugcuau 21180

gcaauauguu uacgaaccca ccuauuacau gcuuaauguu acuagugcug gugaggaugg 21240

uauuucuuau caacccugua cagcuaauug cauugguuau gcugccaaug uauuugcuac 21300

ugagcccaau ggccacauac cagaagguuu uaguuuuaau aauugguuuc uuuuguccaa 21360

ugauuccacu uuggugcaug guaagguggu uuccaaccaa ccauuguugg ucaauugucu 21420

uuuggccauu ccuaagauuu auggacuagg ccaauuuuuc uccuuuaauc aaacgaucga 21480

ugguguuugu aauggagcug cugugcagcg ugcaccagag gcucugaggu uuaauauuaa 21540

ugacaccucu gucauucuug cugaaggcuc aauuguacuu cauacugcuu uaggaacaaa 21600

uuuuucuuuu guuugcagua auuccucaaa uccucacuua gccaccuucg ccauaccucu 21660

gggugcuacc caaguaccuu auuauuguuu ucuuaaagug gauacuuaca acuccacugu 21720

uuauaaauuu uuggcuguuu uaccuccuac cgucagggaa auugucauca ccaaguaugg 21780

ugauguuuau gucaaugggu uuggauacuu gcaucucggu uuguuggaug cugucacaau 21840

uaauuucacu ggucauggca cugacgauga uguuucuggu uuuuggacca uagcaucgac 21900

uaauuuuguu gaugcacuca ucgaaguuca aggaaccgcc auucagcgua uucuuuauug 21960

ugaugauccu guuagccaac ucaaguguuc ucagguugcu uuugaccuug acgaugguuu 22020

uuacccuauu ucuucuagaa accuucugag ucaugaacag ccaauuucuu uuguuacucu 22080

gccaucauuu aaugaucauu cuuuuguuaa cauuacugua ucugcuuccu uuggugguca 22140

uaguggugcc aaccuuauug caucugacac uacuaucaau ggguuuaguu cuuucugugu 22200

ugacacuaga caauuuacca uuucacuguu uuauaacguu acaaacaguu augguuaugu 22260

gucuaaauca caggacagua auugcccuuu caccuugcaa ucuguuaaug auuaccuguc 22320

uuuuagcaaa uuuuguguuu ccaccagccu uuuggcuagu gccuguacca uagaucuuuu 22380

ugguuacccu gaguuuggua gugguguuaa guuuacgucc cuuuauuuuc aauucacaaa 22440

gggugaguug auuacuggca cgacuaaacc acuugaaggu gucacggacg uuucuuuuau 22500

gacucuggau guguguacca aguauacuau cuauggcuuu aaaggugagg guaucauuac 22560

ccuuacaaau ucuagcuuuu uggcaggugu uuauuacaca ucugauucug gacaguuguu 22620

agccuuuaag aaugucacua guggugcugu uuauucuguu acgccauguu cuuuuucaga 22680

gcaggcugca uauguugaug augauauagu ggguguuauu ucuaguuugu cuagcuccac 22740

uuuuaacagu acuagggagu ugccugguuu cuucuaccau ucuaaugaug gcucuaauug 22800

uacagagccu guguuggugu auaguaacau agguguuugu aaaucuggca guauuggcua 22860

cgucccaucu cagucuggcc aagucaagau ugcacccacg guuacuggga auauuaguau 22920

ucccaccaac uuuaguauga guauuaggac agaauauuua cagcuuuaca acacgccugu 22980

uaguguugau ugugccacau auguuuguaa ugguaacucu cguuguaaac aauuacucac 23040

ccaguacacu gcagcaugua agaccauaga gucagcauua caacucagcg cuaggcuuga 23100

gucuguugaa guuaacucua ugcuuacuau uucugaagag gcucuacagu uagcuaccau 23160

uaguucguuu aauggugaug gauauaauuu uacuaaugug cuggguguuu cuguguauga 23220

uccugcaagu ggcagggugg uacaaaaaag gucuuuuauu gaagaccugc uuuuuaauaa 23280

agugguuacu aauggccuug guacuguuga ugaagacuau aagcgcuguu cuaauggucg 23340

cucuguggca gaucuagucu gugcacagua uuacucuggu gucaugguac uaccuggugu 23400

uguugacgcu gagaagcuuc acauguauag ugcgucucuc aucgguggua uggugcuagg 23460

agguuuuacu ucugcagcgg cauugccuuu uagcuaugcu guucaagcua gacucaauua 23520

ucuugcucua cagacggaug uucuacagcg gaaccagcaa uugcuugcug agucuuuuaa 23580

cucugcuauu gguaauauaa cuucagccuu ugagaguguu aaagaggcua uuagucaaac 23640

uuccaagggu uugaacacug uggcucaugc gcuuacuaag guucaagagg uuguuaacuc 23700

gcagggugca gcuuugacuc aacuuaccgu acagcugcaa cacaacuucc aagccauuuc 23760

uaguucuauu gaugacauuu acucucgacu ggacauucuu ucagccgaug uucagguuga 23820

ccgucucauc accggcagau uaucagcacu uaaugcuuuu guugcucaaa cccucacuaa 23880

guauacugag guucaggcua gcaggaaguu agcacagcaa aagguuaaug agugcguuaa 23940

aucgcaaucu cagcguuaug guuuuugugg uggugauggc gagcacauuu ucucucuggu 24000

acaggcagca ccucagggcc ugcuguuuuu acauacagua cuuguaccga gugauuuugu 24060

agauguuauu gccaucgcug gcuuaugcgu uaacgaugaa auugccuuga cucuacguga 24120

gccuggcuua gucuuguuua cgcaugaacu ucaaaaucau acugcgacgg aauauuuugu 24180

uucaucgcga cguauguuug aaccuagaaa accuaccguu agugauuuug uucaaauuga 24240

gaguugugug gucaccuaug ucaauuugac uagagaccaa cuaccagaug uaaucccaga 24300

uuacaucgau guuaacaaaa cacuugauga gauuuuagcu ucucugccca auagaacugg 24360

uccaagucuu ccuuuagaug uuuuuaaugc cacuuaucuu aaucucacug gugaaauugc 24420

agauuuagag cagcguucag agucucuccg uaauacuaca gaggagcucc aaagucuuau 24480

auauaauauc aacaacacac uaguugaccu ugaguggcuc aaccgaguug agacauauau 24540

caaguggccg ugguggguuu gguugauuau uuucauuguu cucaucuuug uugugucauu 24600

acuaguguuc ugcugcauuu ccacggguug uuguggaugc ugcggcugcu gcugugcuug 24660

uuucucaggu uguuguaggg guccuagacu ucaaccuuac gaaguuuuug aaaaggucca 24720

cgugcaguga uguuucuugg acuuuuucaa uacacgauug acacaguugu caaagauguc 24780

ucaaagucug cuaacuuguc uuuggaugcu guccaagagu uggagcucaa uguaguucca 24840

auuagacaag cuucaaaugu gacggguuuu cuuuucacca guguuuuuau cuacuucuuu 24900

gcacuguuua aagcgucuuc uuugaggcgc aauuauauua uguuggcagc gcguuuugcu 24960

gucauuguuc uuuauugccc acuuuuauau uauuguggug cauuuuuaga ugcaacuauu 25020

auuuguugca cacuuauugg caggcuuugu uuagucugcu uuuacuccug gcgcuauaaa 25080

aaugcgcucu uuauuauuuu uaauacuacg acacuuucuu uccucaaugg uaaagcagcu 25140

uauuaugacg gcaaauccau ugugauuuua gaagguggug accauuacau cacuuuuggc 25200

aacucuuuug uugcuuuugu uaguagcauc gacuuguauc uagcuauacg ugggcggcaa 25260

gaagcugacc uacagcuguu gcgaacuguu gagcuucuug auggcaagaa gcuuuauguc 25320

uuuucgcaac aucaaauugu uggcauuacu aaugcugcau uugacucaau ucaacuagac 25380

gaguaugcua caauuaguga augauaaugg ucuaguaguu aauguuauac uuuggcuuuu 25440

cguacucuuu uuccugcuua uuauaagcau uacuuucguc caauugguua aucugugcuu 25500

cacuugucac cgguugugua auagcgcagu uuacacaccu auagggcguu uguauagagu 25560

uuauaagucu uacaugcaaa uagacccccu cccuaguacu guuauugacg uauaaacgaa 25620

auaugucuaa cgguucuauu cccguugaug aggugauuca acaccuuaga aacuggaauu 25680

ucacauggaa uaucauacug acgauacuac uuguagugcu ucaguauggc cauuacaagu 25740

acucugcguu cuuguauggu gucaagaugg cuauucuaug gauacuuugg ccucuugugu 25800

uagcacuguc acuuuuugau gcaugggcua gcuuucaggu caauuggguc uuuuuugcuu 25860

ucagcauccu uauggcuugc aucacucuua ugcuguggau aauguacuuu gucaauagca 25920

uucgguugug gcgcaggaca cauucuuggu ggucuuucaa uccugaaaca gacgcgcuuc 25980

ucacuacuuc ugugaugggc cgacaggucu gcauuccagu gcuuggagca ccaacuggug 26040

uaacgcuaac acuccuuagu gguacauugc uuguagaggg cuauaagguu gcuacuggcg 26100

uacagguaag ucaauuaccu aauuucguca cagucgccaa ggccacuaca acaauugucu 26160

acggacgugu uggucguuca gucaaugcuu caucuggcac ugguugggcu uucuaugucc 26220

gguccaaaca cggcgacuac ucagcuguga guaauccgag uucgguucuc acagauagug 26280

agaaagugcu ucauuuaguc uaaacagaaa cuuuauggcu ucugucaguu uucaggaucg 26340

uggccgcaaa cgggugccau uaucccucua ugccccucuu aggguuacua augacaaacc 26400

ccuuucuaag guacuugcaa auaaugcugu acccacuaau aaaggaaaua aggaccagca 26460

aauuggauac uggaaugagc aaauucgcug gcgcaugcgc cguggugagc gaauugaaca 26520

accuuccaau uggcauuucu acuaccucgg aacaggaccu cacgccgacc uccgcuacag 26580

gacucguacu gaggguguuu ucuggguugc uaaagaaggc gcaaagacug aacccacuaa 26640

ccuggguguc agaaaggcgu cugaaaagcc aauuauucca aauuucucuc aacagcuucc 26700

cagcguaguu gagauuguug aaccuaacac accuccuacu ucacgugcaa auucacguag 26760

caggagucgu gguaauggca acaacagguc cagaucucca aguaacaaca gaggcaauaa 26820

ccagucccgc gguaauucac agaaucgugg aaauaaccag ggucguggag cuucucagaa 26880

cagaggaggc aauaauaaua acaauaacaa gucucguaac caguccaaga acagaaacca 26940

gucaaaugac cgugguggug uaacaucacg cgaugaucug guggcugcug ucaaggaugc 27000

ccuuaaaucu uuggguauug gcgaaaaccc ugacaagcuu aagcaacagc agaagcccaa 27060

acaggaaagg ucugacagca gcggcaaaaa uacaccuaag aagaacaaau ccagagccac 27120

uucgaaagaa cgugaccuca aagacauccc agaguggagg agaauuccca agggcgaaaa 27180

uagcguagca gcuugcuucg gacccagggg aggcuucaaa aauuuuggag augcggaauu 27240

ugucgaaaaa gguguugaug ccucaggcua ugcucagauc gccaguuuag caccaaaugu 27300

ugcagcauug cucuuuggug guaauguggc uguucgugag cuagcggacu cuuacgagau 27360

uacauauaau uauaaaauga cugugccaaa gucugaucca aauguagagc uucuuguuuc 27420

acagguggau gcauuuaaaa cugggaaugc aaaaccccag agaaagaagg aaaagaagaa 27480

caagcgugaa accacgcagc agcugaauga agaggccauc uacgaugaug ugggugugcc 27540

aucugaugug acucaugcca auuuggaaug ggacacagcu guugauggug gugacacggc 27600

cguugaaauu aucaacgaga ucuucgacac aggaaauuaa acaauguuug acuggcuuau 27660

ccuggcuaug ucccagggua gugccauuac acuguuauua cugaguguuu uucuagcgac 27720

uuggcugcug ggcuauggcu uugcccucua acuagcgguc uuggucuugc acacaacggu 27780

aagccagugg uaaugucagu gcaagaagga uauuaccaua gcacugucau gaggggaacg 27840

caguaccuuu ucaucuaaac cuuugcacga guaaucaaag auccgcuuga cgagccuaua 27900

uggaagagcg ugccagguau uugacucaag gacuguuagu aacugaagac cugacggugu 27960

<210> 33

<211> 27976

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV 1251-125-10 (125-10) passage30 Genomic cDNA

<400> 33

aattcaacta aacgaaattt tgtccttccg gccgcatgtc catgctgctg gaagctgacg 60

tggaatttca ttaggtttgc ttaagtagcc atcgcaagtg ctgtgctgtc ctctagttcc 120

tggttggcgt tccgtcgcct tctacatact agacaaacag ccttcctccg gttccgtctg 180

ggggttgtgt ggataactag ttccgtctag tttgaaacca gtaactgtcg gctatggcta 240

gcaaccatgt tacattggct tttgccaatg atgcagaaat ttcagctttt ggcttttgca 300

ctgctagtga agccgtctca tactattctg aggccgccgc tagtggattt atgcaatgcc 360

gtttcgtgtc cttcgatctc gctgacactg ttgagggatt gcttcccgaa gactatgtca 420

tggtggtggt cggcactacc aagcttagtg cgtatgtgga cacttttggt agccgcccca 480

aaaacatttg tggttggctg ttattttcta actgtaatta cttcctcgaa gagttagagc 540

ttacttttgg tcgtcgtggt ggtaacatcg tgccagttga ccaatacatg tgtggcgctg 600

acggtaaacc tgttcttcag gaatccgaat gggagtatac agatttcttt gctgactccg 660

aagacggtca actcaacatt gctggtatca cttatgtgaa ggcctggatt gtagagcgat 720

cggatgtctc ttatgcgagt cagaatttaa catctattaa gtctattact tactgttcaa 780

cctatgagca tacttttcct gatggtactg ccatgaaggt tgcacgtact ccaaagatta 840

agaagactgt tgtcttgtct gagccacttg ctactatcta cagggaaatt ggttctcctt 900

ttgtggataa tgggagcgat gctcgttcta tcattaagag accagtgttc ctccacgctt 960

ttgttaagtg taagtgtggt agttatcatt ggactgttgg tgattggact tcctatgtct 1020

ccacttgctg tggctttaag tgtaagccag tccttgtggc ttcatgctct gctacgcctg 1080

gttctgttgt ggttacgcgc gctggtgctg gcactggtgt taagtattac aacaacatgt 1140

tcctgcgcca tgtggcagac attgatgggt tggcattctg gcgaattctc aaggtgcagt 1200

ccaaagacga cctcgcttgc tctggtaaat tccttgaaca ccatgaggaa ggtttcacag 1260

atccttgcta ctttttgaat gactcgagca ttgctactaa gctcaagttt gacatcctta 1320

gtggcaagtt ttctgatgaa gtcaaacaag ctatctttgc tggtcatgtt gttgttggca 1380

gcgcgmtcgt tgacattgtt gacgatgcac tgggacagcc ttggtttata cgtaagcttg 1440

gtgaccttgc aagtgcagct tgggagcagc ttaaggctgt cgttagaggc cttaacctcc 1500

tgtctgatga ggtcgtgctc tttggcaaaa gacttagctg tgccactctt agtatcgtta 1560

acggtgtttt tgagttcatc gccgaagtgc ctgagaagtt ggctgcggct gttacagttt 1620

ttgtcaactt cttgaatgag ctttttgagt ctgcctgtga ctgcttaaag gtcggaggta 1680

aaacctttaa caaggttggc tcttatgttc tttttgacaa cgcattggtt aagcttgtca 1740

aggcaaaagt tcgcggccca cgacaggcag gtgtttgtga agttcgttac acaagccttg 1800

ttattgggag tactaccaag gtggtttcca agcgcgttga aaatgccaat gtgaatctcg 1860

tcgtcgttga cgaggatgtg accctcaaca ccactggtcg tacagttgtt gttgacggac 1920

ttgcattctt cgagagtgac gggttttaca gacatcttgc tgatgctgac gttgtcattg 1980

aacatcctgt ttataagtct gcttgtgagc tcaagccagt ttttgagtgt gacccaatac 2040

ctgattttcc tatgcctgtg gccgctagtg ttgcagagct ttgtgtgcaa actgatctgt 2100

tgcttaaaaa ttacaacact ccttataaaa cttacagctg cgttgtgaga ggtgataagt 2160

gttgtatcac ttgcacctta catttcacag caccaagtta tatggaggct gctgctaatt 2220

ttgtagacct ctgtaccaag aacattggta ctgctggttt tcatgagttt tacattacgg 2280

cccatgaaca acaggatctg caagggttcg taaccacttg ttgcacgatg tcaggttttg 2340

agtgttttat gcctataatc ccacagtgtc cagcagtgct tgaagagatt gatggtggta 2400

gcatctggcg gtcttttatc actggtctta atacaatgtg ggatttttgc aagcatctta 2460

aagtcagctt tggactagat ggcattgttg tcactgtagc acgcaaattt aaacgacttg 2520

gtgctctctt ggcagaaatg tataacactt acctttcaac tgtggtggaa aacttggtac 2580

tggccggtgt tagcttcaag tattatgcca ccagtgtccc aaaaattgtt ttgggctgtt 2640

gttttcacag tgttaaaagt gttcttgcaa gtgccttcca gattcctgtc caggcaggcg 2700

ttgagaagtt taaagtcttc cttaactgtg ttcaccctgt tgtaccacgt gtcattgaaa 2760

cttcttttgt ggaattagaa gagacgacat ttaaaccacc agcactcaat ggtagtattg 2820

ctattgttga tggctttgct ttctattatg atggaacact atactatccc accgatggta 2880

atagcgttgt tcctatctgc tttaagaaga aaggtggtgg tgatgtcaaa ttctctgatg 2940

aagtctctgt taaaaccatt gacccagttt ataaggtctc ccttgaattt gagttcgagt 3000

ctgagactat tatggctgtg cttaataagg ctgttggtaa ttgtatcaag gttacaggtg 3060

gttgggacga tgttgttgag tatatcaatg ttgccattga ggttcttaaa gatcacatcg 3120

atgtgcctaa gtactacatc tatgatgagg aaggtggcac cgatcctaat ctgcccgtaa 3180

tggtttctca gtggccgttg aatgatgaca cgatctcaca ggatctgctt gatgttgaag 3240

ttgttactga tgcgccagtt gatttcgagg gtgatgaagt agactcctct gaccctgwta 3300

aggtggcaga cgtggctaac tctgagcctg aggatgacgg tcttaatgta gctcctgaaa 3360

caaatgtaga gtctgaagtt gaggaagttg ccgcaacctt gtcctttaaa gatacacctt 3420

ccacagttac taaggatcct tttgcttttg actttgcaag ctatggagga cttaaggttt 3480

taagacaatc tcataacaac tgctgggtta cttctacctt ggtgcagcta caattgcttg 3540

gcatcgttga tgaccctgca atggagcttt ttagtgctgg tagagttggt ccaatggttc 3600

gcaaatgcta tgagtcacaa aaggctatct tgggatcttt gggtgatgtg tcggcttgcc 3660

tagagtctct gactaaggac ctacacacac ttaagattac ctgttctgta gtctgtggtt 3720

gtggtactgg tgaacgtatc tatgatggtt gtgcttttcg tatgacgcca actttggaac 3780

cgttcccata tggtgcttgt gctcagtgtg ctcaagtttt gatgcacact tttaaaagta 3840

ttgttggcac cggcatcttt tgtcgagata ctactgctct ctccttggat tctttggttg 3900

taaaacctct ttgtgcggct gcttttatag gcaaggatag tggtcattat gtcactaact 3960

tttatgatgc tgctatggct attgatggtt atggtcgtca tcagataaag tatgacacac 4020

tgaacactat ttgtgttaaa gacgttaatt ggacagcacc ttttgtccca gacgttgagc 4080

ctgtattgga gcctgttgtc aaacctttct attcttataa gaatgttgat ttttaccaag 4140

gagattttag tgaccttgtt aaacttccat gtgattttgt tgttaatgct gcaaatgaga 4200

atttgtctca cggtggcggc atagcaaagg ccattgatgt ttataccaag ggcatgttgc 4260

agaagtgctc gaatgattac attaaagcac acggtcccat taaagttgga cgtggtgtca 4320

tgttggaggc attaggtctt aaggtcttta atgttgttgg tccacgtaag ggtaagcatg 4380

cacctgagct tcttgttaag gcttataagt ccgtttttgc taattcaggt gttgctctta 4440

cacctttgat tagtgttgga atttttagtg ttcctttgga agaatcttta tctgcttttc 4500

ttgcatgtgt tggtgatcgc cactgtaagt gcttttgtta tagtgacaaa gagcgcgagg 4560

cgatcattaa ttacatggat ggcttggtag atgctatttt caaagatgca cttgttgata 4620

ctactcctgt ccaggaagat gttcaacaag tttcacaaaa accagttttg cctaattttg 4680

aacctttcag gattgaaggt gctcatgctt tctatgagtg caaccctgaa ggtttgatgt 4740

cattaggtgc tgacaagctg gtgttgttta caaattccaa tttggatttt tgtagcgttg 4800

gtaagtgtct taacaatgtg actggcggtg cattgcttga agccataaat gtatttaaaa 4860

agagtaacaa aacagtgcct gctggcaact gtgttacttt tgagtgtgca gatatgattt 4920

ctattactat ggtagtattg ccatctgacg gtgatgctaa ttatgacaaa aattatgcac 4980

gcgccgtcgt caaggtatct aagcttaaag gcaagttatt gcttgctgtt ggtgatgcca 5040

tgttgtattc caagttgtcc cacctcagcg tgttaggttt cgtatccaca cctgatgatg 5100

tggagcgttt ctacgcaaat aagagtgtgg ttattaaagt tactgaggat acacgtagtg 5160

ttaagactgt taaagtagaa tccactgtta cttatggaca acaaattgga ccttgtcttg 5220

ttaatgacac cgttgtcaca gacaacaaac ctgttgttgc tgatgttgta gctaaggttg 5280

taccaagtgc taattgggat tcacattatg gttttgataa ggctggtgag ttccacatgc 5340

tagaccatac tgggtttgcc tttcctagtg aagttgttaa cggtaggcgt gtgcttaaaa 5400

ccacagataa taactgttgg gttaatgtta catgtttaca attacagttt gctagattta 5460

ggttcaagtc agcaggtcta caggctatgt gggagtccta ttgtactggt gatgttgcta 5520

tgtttgtgca ttggttgtac tggcttactg gtgttgacaa aggtcagcct agtgattcag 5580

aaaatgcact taacatgttg tctaagtaca ttgttcctgc tggttctgtc actattgaac 5640

gtgtcacgca tgacggttgt tgttgtagta agcgtgttgt cactgcacca gttgtgaatg 5700

ctagcgtgtt gaagcttggc gtcgaggatg gtctttgtcc acatggtctt aactacattg 5760

acaaagttgt tgtagttaaa ggtactacaa ttgttgtcaa tgttggaaaa cctgtagtgg 5820

caccatcgca cctctttctt aagggtgttt cctacacaac attcctagat aatggtaacg 5880

gtgttgccgg ccattatact gtttttgatc atgacactgg tatggtgcat gatggagatg 5940

tttttgtacc aggtgatctc aatgtgtctc ctgttacaaa tgttgtcgtc tcagagcaga 6000

cggctgttgt gattaaagac cctgtgaaga aagtagagtt agacgctaca aagctgttag 6060

acactatgaa ttatgcatcg gaaagattct tttcctttgg tgattttatg tcacgtaatt 6120

taattacagt gtttttgtac atccttagta ttttgggtct ctgttttagg gcctttcgta 6180

agagggatgt taaagttcta gctggtgtac cccaacgtac tggtattata ttgcgtaaaa 6240

gtgtgcgcta taatgcaaag gctttgggtg tcttcttcaa gctaaaactt tattggttca 6300

aagttcttgg taagtttagt ttgggtattt atgcattgta tgcattacta ttcatgacaa 6360

tacgctttac acctataggt ggccctgttt gtgatgatgt tgttgctggt tatgctaatt 6420

ctagttttga caagaatgag tattgcaaca gtgttatttg taaggtctgt ctctatgggt 6480

accaggaact ttcggacttc tctcacacac aggtagtatg gcaacacctt agagacccat 6540

taattggtaa tgtgatgcct ttcttttatt tggcatttct ggcaattttt gggggtgttt 6600

atgtaaaggc tattactctc tattttattt tccagtatct taacatactt ggtgtgtttt 6660

tgggcctaca acagtccatt tggtttttgc agcttgtgcc ttttgatgtc tttggtgacg 6720

agatcgtcgt ctttttcatc gttacacgcg tattgatgtt ccttaagcat gttttccttg 6780

gctgcgataa ggcatcttgt gtggcttgct ctaagagtgc tcgccttaag cgcgttcctg 6840

tccagactat ttttcagggt actagcaaat ccttctacgt acatgccaat ggtggttcta 6900

agttctgtaa gaagcacaat ttcttttgtt taaattgtga ttcttatggt ccaggctgca 6960

cttttattaa tgacgtcatt gcaactgaag ttggtaatgt tgtcaaactt aatgtgcaac 7020

cgacaggtcc tgccactatt cttattgaca aggttgaatt cagtaatggt ttttactatc 7080

tttatagtgg tgacacattt tggaagtaca actttgacat aacagataac aaatacactt 7140

gcaaagagtc acttaaaaat tgtagcataa tcacagactt tattgttttt aacaataatg 7200

gttccaatgt aaatcaggtt aagaatgcat gtgtgtattt ttcacagatg ctttgtaaac 7260

ctgttaagtt agtggactca gcgttgttgg ccagtttgtc tgttgatttt ggtgcaagct 7320

tacatagtgc ttttgttagt gtgttgtcga atagttttgg caaagacctg tcaagttgta 7380

atgacatgca ggattgcaag agcacattgg gttttgatga tgtaccattg gataccttta 7440

atgctgctgt tgctgaggct catcgttacg atgtcctctt gactgacatg tcgttcaaca 7500

attttaccac cagttatgca aaaccagagg aaaaacttcc cgtccatgac attgccacgt 7560

gtatgcgtgt aggtgccaag attgttaatc ataacgttct tgtcaaggat agtatacctg 7620

tggtgtggct tgtacgtgat ttcattgccc tttctgaaga aactaggaag tacattattc 7680

gtacgactaa agttaagggt ataaccttca tgttgacctt taatgattgt cgtatgcata 7740

ctaccatacc tactgtttgc attgcaaata agaagggtgc aggtcttcct agtttttcaa 7800

aggttaagaa attcttctgg tttttgtgtc tgttcatagt tgctgttttc tttgcactaa 7860

gcttttttga ttttagtact caggttagca gtgatagtga ttatgacttc aagtatattg 7920

agagtggcca gttgaagact tttgacaatc cacttagttg tgtgcataat gtctttagta 7980

acttcgacca gtggcatgat gccaagtttg gtttcacccc cgtcaacaat cctagttgtc 8040

ctatagtcgt tggtgtatca gacgaagcgc gcactgttcc aggtatccca gcaggtgttt 8100

atttagctgg taaaacactt gtttttgcta ttaacaccat ttttggtaca tctggtttgt 8160

gctttgatgc tagtggcgtt gctgataagg gcgcttgcat ttttaattcg gcttgcacca 8220

cattatctgg tttgggtgga actgctgtct actgttataa gaatggtcta gttgaaggtg 8280

ctaaacttta tagtgagttg gcacctcata gctactataa aatggtagat ggtaatgctg 8340

tgtctttacc tgaaattatc tcacgcggct ttggcatccg tactatccgt acaaaggcta 8400

tgacctactg tcgcgttggc cagtgtgtgc aatctgcaga aggtgtttgt tttggcgccg 8460

atagattctt tgtctataat gcagaatctg gttctgactt tgtttgtggc acagggctct 8520

ttacattgtt gatgaacgtt attagtgttt tttccaagac agtaccagta actgtgttgt 8580

ctggtcaaat actttttaat tgcattattg cttttgctgc tgttgcggtg tgtttcttat 8640

ttacaaagtt taagcgcatg ttcggtgata tgtctgttgg cgttttcact gtcggtgctt 8700

gtactttgtt gaacaatgtt tcctacattg taacacagaa cacacttggc atgttgggct 8760

atgcaacttt gtactttttg tgcactaaag gtgttagata tatgtggatt tggcatttgg 8820

gatttttgat ctcatatata cttattgcac catggtgggt tttgatggtt tatgcctttt 8880

cagccatttt tgagtttatg cctaaccttt ttaagcttaa ggtttcaaca caactttttg 8940

agggtgacaa gttcgtaggc tcttttgaaa atgctgcagc aggtacattt gtgcttgata 9000

tgcatgccta tgagagactt gccaactcta tctcaactga aaaactgcgt cagtatgcta 9060

gtacttacaa taagtacaag tattattcag gcagtgcttc agaggctgat tacaggcttg 9120

cttgttttgc ccatttggcc aaggctatga tggattatgc ttctaatcac aacgacacgt 9180

tatacacacc acccactgtg agttacaatt caactctaca ggctggcttg cgtaagatgg 9240

cacaaccatc tggtgttgtt gagaagtgca tagttcgtgt ttgctatggt aatatggctc 9300

ttaatggcct atggcttggt gatactgtta tctgcccacg ccatgttata gcgtctagta 9360

ctactagcac tatagattat gactatgccc tttctgtttt acgcctcyac aacttctcca 9420

tttcatctgg taatgttttc ctaggtgttg tgggtgtaac catgcgaggt gctttgttgc 9480

agataaaggt taatcaaaac aatgtccaca cgcctaagta cacctatcgc acagttagac 9540

cgggtgaatc ttttaatatc ttggcgtgct atgatggttc tgcagctggt gtttacggcg 9600

ttaacatgcg ctctaattac actattagag gctcgttcat taatggcgct tgtggttcac 9660

ctggttataa cattaacaat ggtaccgttg agttttgcta tttacaccag cttgaacttg 9720

gttcaggctg tcatgttggt agcgacttag atggtgttat gtatggtggt tatgaggacc 9780

aacctacttt gcaagttgaa ggcgctagta gtctgtttac agagaatgtg ttggcatttc 9840

tttatgcagc actcattaat ggttctacct ggtggcttag ttcttctagg attgctgtag 9900

acaggtttaa tgagtgggct gttcataatg gtatgacaac agtagttaat actgattgct 9960

tttctattct tgctgctaag actggtgttg atgtacaacg tttgttggcc tcaatccagt 10020

ctctgcataa gaattttggt ggaaagcaaa ttcttggcta tacctcgttg acagatgagt 10080

ttactacagg tgaagttata cgtcaaatgt atggcgttwa tcttcagagt ggttatgttt 10140

cacgcgcctg tagaaatgtc ttgctggttg gttcttttct gactttcttt tggtcagaat 10200

tagtttccta cactaagttc ttttgggtaa atcctggtta tgtcacacct atgtttgcgt 10260

gtttgtcatt gctgtcctca cttttgatgt tcacactcaa gcataagaca ttgtttttcc 10320

aggtctttct aatacctgct ctgattgtta catcttgcat taatttggca tttgatgttg 10380

aagtctacaa ctatttggca gagcattttg attaccatgt ttctctcatg ggttttaatg 10440

cacaaggtct tgttaacatc tttgtctgct ttgttgttac cattttacac ggcacataca 10500

catggcgctt ttttaacaca cctgtgagtt ctgtcactta tgtggtagct ttgctgactg 10560

cggcatataa ctatttttac gctagtgaca ttcttagttg tgctatgaca ctatttgcta 10620

gtgtgactgg caactggttc gttggtgctg tttgttataa agctgctgtt tatatggcct 10680

tgagatttcc tacttttgtg gctatttttg gtgatattaa gagtgttatg ttctgttacc 10740

ttgtgttggg ttattttacc tgttgcttct acggtattct ctactggttc aacaggtttt 10800

ttaaggttag tgtaggtgtc tatgactata ctgttagtgc tgctgagttt aagtatatgg 10860

ttgctaacgg cctacgtgca ccaactggaa cacttgattc actacttctg tctgccaaat 10920

tgattggtat tggtggtgag cggaatatta agatttcttc cgttcagtct aaactgactg 10980

atattaagtg tagtaacgtt gtgcttttag gctgtctctc tagcatgaat gtctcagcaa 11040

attcaacaga atgggcctat tgtgttgact tgcataacaa gatcaacttg tgtaatgacc 11100

cagaaaaagc gcaggaaatg ctacttgctt tgttggcatt tttccttagt aagaatagtg 11160

cttttggttt agatgactta ttggaatcct attttaatga caatagtatg ttgcagagtg 11220

ttgcatctac ttatgtcggt ttgccttctt atgtcattta tgaaaatgca cgccaacagt 11280

atgaagatgc tgttaataat ggttctccac ctcagttggt taagcaattg cgccatgcca 11340

tgaatgtagc aaagagcgaa tttgaccgtg aggcttctac tcagcgtaag cttgatagaa 11400

tggcggaaca ggctgcagca cagatgtaca aagaggcacg agcagttaat aggaagtcca 11460

aagttgtaag tgctatgcat tcactgcttt ttggtatgtt gagacgtttg gacatgtctt 11520

ctgtagacac cattctcaac ttggcaaagg atggggttgt acctctgtct gtcataccgg 11580

cagtcagtgc tactaagctt aacattgtta cttctgatat cgattcttat aatcgtatcc 11640

agcgtgaggg atgtgtccac tacgctggta ccatttggaa tataattgat atcaaggaca 11700

atgatggcaa ggtggtacac gttaaggagg taaccgcaca gaatgctgag tccctgtcat 11760

ggcccctggt ccttgggtgt gagcgtattg tcaagctcca gaataatgaa attattcctg 11820

gtaagctgaa gcagcgctcc attaaggcag aaggagatgg catagttgga gaaggtaagg 11880

cactttacaa taatgagggt ggacgtactt ttatgtatgc tttcatctcg gacaaaccgg 11940

acctgcgtgt agtcaagtgg gagttcgatg gtggttgtaa cactattgag ctagaaccac 12000

cacgtaagtt cttggtggat tctcctaatg gtgcacagat caagtatctc tactttgttc 12060

gtaaccttaa cacgttacgt aggggtgctg ttctcggcta cataggtgcc actgtacgct 12120

tgcaggctgg taaacaaaca gaacaggcta ttaactcttc attgttgaca ctttgcgctt 12180

tcgctgtgga tcctgctaag acctacatcg atgctgtcaa aagtggtcac aaaccagtag 12240

gtaactgtgt taagatgttg gccaatggtt ctggtaatgg acaagctgtt actaatggtg 12300

tggaggctag tactaaccag gattcatacg gtggtgcgtc cgtgtgtcta tattgtagag 12360

cacatgttga gcatccatct atggatggtt tttgcagact gaaaggcaag tacgtacagg 12420

ttccactagg tacagtggat cctatacgtt ttgtacttga gaatgacgtt tgcaaggttt 12480

gtggttgttg gctggctaat ggctgcactt gtgacagatc cattatgcaa agcactgata 12540

tggcttattt aaacgagtac ggggctctag tgcagctcga ctagagccct gtaacggtac 12600

tgatacacaa catgtgtatc gtgcttttga catctacaac aaggatgttg cttgtctagg 12660

taaattcctc aaggtgaact gtgttcgcct gaagaatttg gataagcatg atgcattcta 12720

tgttgtcaaa agatgtacca agtctgcgat ggaacacgag caatccatct atagcagact 12780

tgaaaagtgt ggagccgtag ccgaacacga tttcttcact tggaaggatg gtcgtgccat 12840

ctatggtaac gtttgtagaa aggatcttac cgagtatact atgatggatt tgtgttacgc 12900

tttacgtaac tttgatgaaa acaattgcga tgttcttaag agcattttaa ttaaggtagg 12960

cgcttgtgag gagtcctact tcaataataa agtctggttt gaccctgttg aaaatgaaga 13020

cattcatcgt gtctatgcat tgttaggtac cattgtttca cgtgctatgc ttaaatgcgt 13080

taagttctgt gatgcaatgg ttgaacaagg tatagttggt gttgtcacat tagataatca 13140

ggatcttaat ggtgattttt atgattttgg tgattttact tgtagcatca agggaatggg 13200

tatacccatt tgcacatcat attactctta tatgatgcct gttatgggta tgactaattg 13260

ccttgctagt gagtgttttg ttaagagtga tatatttggt gaggatttca agtcatatga 13320

cctgctggaa tatgatttca cggagcataa gacagcactc ttcaacaagt atttcaagta 13380

ttggggactg caataccacc ctaactgtgt ggactgcagt gatgagcagt gcatagttca 13440

ctgtgccaac ttcaatacgt tgttttccac tactatacct attacggcat ttggaccttt 13500

gtgtcgcaag tgttggattg atggtgttcc actggtaact acagctggtt atcattttaa 13560

acagttaggt atagtttgga acaatgacct caacttacac tctagcaggc tctctattaa 13620

cgaattactc cagttttgta gtgatcctgc attgcttata gcatcatcac cagcccttgt 13680

tgatcagcgt actgtttgct tttcagttgc agcgctaggt acaggtatga ctaaccagac 13740

tgttaaacct ggccatttca ataaggagtt ttatgacttc ttacttgagc aaggtttctt 13800

ttctgagggc tctgagctta ctttaaagca cttcttcttt gcacagaagg gtgatgcagc 13860

tgttaaggat tttgactact ataggtataa tagacctact gttctggaca tttgccaagc 13920

tcgcgtcgtg tatcaaatag tgcaacgcta ttttgatatt tacgaaggtg gttgtatcac 13980

tgctaaagag gtggttgtta caaaccttaa caagagcgca ggttatcctt tgaacaagtt 14040

tggtaaagct ggtctttact atgagtcttt atcctatgag gaacaggatg aactttatgc 14100

ttatactaag cgtaacatcc tgcccactat gacacagctc aaccttaaat atgctataag 14160

tggcaaagaa cgtgcacgca cagtgggtgg tgtttcgctt ttgtcaacca tgactactcg 14220

gcagtatcat cagaaacacc ttaagtccat agttaatact aggggcgctt cggttgttat 14280

tggtactact aagttttatg gtggttggga caatatgctt aagaacctta ttgatggtgt 14340

tgaaaatccg tgtcttatgg gttgggacta cccaaagtgc gacagagcac tgcccaatrt 14400

gatacgtatg atttcagcca tgattttagg ctctaagcac accacatgct gcagttccac 14460

tgaccgcttt ttcaggttgt gcaatgaatt ggctcaagtc cttactgagg ttgtttattc 14520

taatggaggt ttttatttga agccaggtgg tactacctct ggtgatgcaa ccaccgcata 14580

tgcaaactca gtttttaata tcttccaagc agtaagtgcc aatgttaaca aacttcttag 14640

tgttgacagc aatgtctgtc ataatttaga agttaagcaa ttgcagcgta agctttatga 14700

gtgctgttat agatcaacta ccgtcgatga ccagttcgtc gttgagtatt atggttactt 14760

gcgtaaacat ttttcaatga tgattctttc tgatgatggc gttgtttgtt ataacaatga 14820

ctatgcatca cttggttatg tcgctgatct taacgcattc aaggctgttt tgtattacca 14880

gaacaatgtc ttcatgagcg cctctaaatg ttggatcgag cctgacatta ataaaggtcc 14940

tcatgaattt tgctcgcagc atactatgca gattgtcgat aaagatggta cttattacct 15000

tccttaccct gatccttcaa gaattctctc tgcaggtgtg tttgttgatg acgttgttaa 15060

aactgatgca gttgtattgc ttgaacgtta tgtgtcattg gctatagatg cctacccgtt 15120

atctaagcat gaaaaccctg aatataagaa ggtgttttat gtgcttttgg attgggttaa 15180

gcatctgtac aaaactctta atgctggtgt gttagagtct ttttctgtca cacttttgga 15240

agattctact gctaaattct gggatgagag cttttatgcc aacatgtatg agaaatctgc 15300

agttttacaa tctgcagggc tttgtgttgt ttgtggctct caaactgttt tacgttgtgg 15360

tgattgtcta cggcgtccta tgctttgtac taagtgtgct tatgatcatg tcattggaac 15420

aactcacaag ttcattttgg ccatcactcc atatgtgtgt tgtgcttcag attgtggtgt 15480

caatgatgta actaagctct acttaggtgg tcttagttat tggtgtcatg accacaagcc 15540

acgtcttgca ttcccgttgt gctctgctgg taatgttttt ggcttgtaca aaaattctgc 15600

taccggctca cccgatgttg aagactttaa tcgcattgct acatccgatt ggactgatgt 15660

ttctgactac aggttggcaa atgatgtcaa ggactcattg cgtctgtttg cagcggaaac 15720

tatcaaggcc aaggaggaga gcgttaagtc atcctatgct tgtgcaacac tacatgaggt 15780

tgtaggacct aaagagttgt tgctcaaatg ggaagtcggc agacccaaac caccccttaa 15840

tagaaattcg gttttcactt gttatcatat aacgaagaac accaaatttc aaatcggtga 15900

gtttgtgttt gagaaggcag aatatgataa tgatgctgta acatataaaa ctaccgccac 15960

aacaaaactt gttcctggca tggtttttgt gcttacctca cataatgttc agccattgcg 16020

cgcaccgacc attgctaatc aagaacgtta ttccactata cataagttgc atcctgcttt 16080

taacatacct gaagcttatt ctagcttagt gccctattac caattgattg gtaagcagaa 16140

gattacaact attcagggac ctcccggtag tggtaaatct cactgtgtta tagggctagg 16200

tttgtactat ccaggtgcac gtatagtgtt tacagcttgt tctcatgcag cggtcgattc 16260

actttgtgtg aaagcttcca ctgcttatag caatgacaaa tgttcacgca tcataccaca 16320

gcgcgctcgt gttgagtgtt atgatggttt caagtctaat aatactagtg ctcagtacct 16380

tttctctact gtcaatgctt tgccagagtg caatgcggac attgttgtgg tggatgaggt 16440

ctctatgtgc actaattatg acttgtctgt cataaatcag cgcatcagct ataggcatgt 16500

agtctatgtt ggtgaccctc aacagctgcc tgcaccacgt gttatgattt cacgtggtac 16560

tttggaacca aaggactaca acgttgtcac tcaacgcatg tgtgccctta agcctgatgt 16620

tttcttgcac aagtgttatc gctgtcctgc tgagatagtg cgtactgtgt ctgagatggt 16680

ctatgaaaac caattcattc ctgtgcaccc agatagcaag cagtgtttta aaatcttttg 16740

caagggtaat gttcaggttg ataatggttc aagcattaat cgcaggcaat tggatgttgt 16800

gcgtatgttt ttggctaaaa atcctaggtg gtcaaaggct gtttttattt ctccttataa 16860

cagccagaat tatgttgcca gccgcatgct aggtctacaa attcagacag ttgactcatc 16920

ccagggtagt gagtatgact atgtcattta cacacaaact tcagatactg cccatgcctg 16980

taatgttaac aggtttaatg ttgccatcac aagggccaag aaaggcatat tatgtataat 17040

gtgcgatagg tccctttttg atgtgcttaa attctttgag cttaaattgt ctgatttgca 17100

ggctaatgag ggttgtggtc tttttaaaga ctgtagcaga ggtgatgatc tgttgccacc 17160

atctcacgct aacaccttca tgtctttagc ggacaatttt aagactgatc aagatcttgc 17220

tgttcaaata ggtgttaatg gacccattaa atatgagcat gttatctcgt ttatgggttt 17280

ccgttttgat atcaacatac ccaaccatca tactctcttt tgcacacgcg actttgccat 17340

gcgcaatgtt agaggttggt taggctttga cgttgaagga gcacatgttg ttggctctaa 17400

cgtcggtaca aatgtcccat tgcaattagg gttttctaac ggtgttgatt ttgttgtcag 17460

acctgaaggt tgcgttgtaa cagagtctgg tgactacatt aaacccgtca gagctcgtgc 17520

tccaccaggg gaacaattcg cacacctttt gcctttactt aaacgcggcc aaccatggga 17580

tgttgtccgc aaacgtatag tgcagatgtg tagtgactac ctggccaacc tatcagacat 17640

actaattttt gtgttgtggg ctggtggttt ggagttgaca actatgcgtt attttgtcaa 17700

gattggacca agtaagagtt gtgattgtgg taaggttgct acttgttaca atagtgcgct 17760

gcatacgtac tgttgtttca aacatgccct tggttgtgat tatctgtata acccatactg 17820

tattgatata cagcagtggg gatacaaggg atcacttagc cttaaccacc atgagcattg 17880

taatgtacat agaaacgagc atgtggcttc tggtgatgcc ataatgactc gctgtctggc 17940

catacatgat tgctttgtca agaacgttga ctggtccatc acatacccat ttattggtaa 18000

tgaggctgtt attaataaga gcggccgaat tgtgcaatca cacactatgc ggtcagttct 18060

taagttatac aatccgaaag ccatatatga tattggcaat cctaagggca ttagatgtgc 18120

cgtaacggat gctaagtggt tttgctttga caagaatcct aytaattcta atgtcaagac 18180

attggagtat gactatataa cacatggcca atttgatggg ttgtgcttgt tttggaattg 18240

caatgtagac atgtatccag aattttctgt ggtctgtcgt tttgatactc gctgtaggtc 18300

accactcaac ttggagggtt gtaatggtgg ttcactgtat gttaataatc atgcattcca 18360

tacaccggct tttgacaagc gtgcttttgc taagttgaag ccaatgccat ttttctttta 18420

tgatgatact gagtgtgaca agttacagga ctccataaac tatgttcctc ttagggctag 18480

taactgcatt actaaatgta atgttggtgg tgctgtctgt agtaagcatt gtgctatgta 18540

tcatagctat gttaatgctt acaacacttt tacgtcggcg ggctttacta tttgggtgcc 18600

tacttcgttt gacacctata atctgtggca gacatttagt aacaatttgc aaggtcttga 18660

gaacattgct ttcaatgtcg taaagaaagg atcttttgtt ggtgccgaag gtgaacttcc 18720

tgtagctgtg gttaatgaca aagtgctcgt tagagatggt actgttgata ctcttgtttt 18780

tacaaacaag acatcactac ccactaacgt agcttttgag ttgtatgcca agcgtaaggt 18840

aggactcacc ccacccatta cgatcctacg taacttgggt gtagtttgta catctaagtg 18900

tgtcatttgg gactatgaag ccgaacgtcc acttactact tttacaaagg atgtttgtaa 18960

atataccgac tttgagggtg acgtctgtac actctttgat aacagcattg ttggttcatt 19020

agagcgattc tccatgaccc aaaatgctgt gcttatgtca cttacagctg ttaaaaagct 19080

taytggcata aagttaactt atggttatct taatggtgtc ccagttaaca cacatgaaga 19140

taaacctttt acttggtata tttacactag gaagaacggc aagttcgagg accatcctga 19200

tggctatttt acccaaggta gaacaaccgc tgattttagc cctcgtagcg acatggaaaa 19260

ggacttccta agtatggata tgggtctgtt tattaacaag tacggacttg aagattacgg 19320

ctttgagcac gttgtgtatg gtgatgtttc aaaaaccacc cttggtggtt tgcatctact 19380

aatttcgcag gtgcgtctgg cctgtatggg tgtgctcaaa atagacgagt ttgtgtctag 19440

taatgatagc acgttaaagt cttgtactgt tacatatgct gataacccta gtagtaagat 19500

ggtttgtacg tatatggatc tcctgcttga cgattttgtc agcattctta aatctttgga 19560

tttgggcgtt gtatctaaag ttcatgaagt tatggtcgat tgtaaaatgt ggaggtggat 19620

gttgtggtgt aaggatcata aactccagac attttatccg caacttcagg ccagtgaatg 19680

gaagtgtggt tattccatgc cttctattta caagatacaa cgtatgtgtt tagaaccttg 19740

caatctctac aactatggtg ctggtattaa gttacctgat ggcattatgt ttaacgtagt 19800

taaatacaca cagctttgtc aatatctcaa tagcaccaca atgtgtgtac cccatcacat 19860

gcgtgtgcta catcttggtg ctggctccga caagggtgtt gcacctggca cggctgtctt 19920

acgacgttgg ttgccactgg atgccattat agttgacaat gatagtgtgg attacgttag 19980

cgatgctgat tatagtgtta caggagattg ctctacctta tacctgtcag ataagtttga 20040

tttagttata tctgatatgt atgatggtaa gattaaaagt tgtgatgggg agaacgtgtc 20100

taaagaaggc ttctttccct atattaatgg tgtcatcacc gaaaagttgg cacttggtgg 20160

tactgtagct attaaggtga cggagtttag ttggaataag aagttgtatg aactcattca 20220

gaggtttgag tattggacaa tgttctgtac cagtgttaac acgtcatcgt cagaggcatt 20280

cttaattggt gttcactatt taggtgattt tgcaagtggc gctgtgattg acggcaacac 20340

tatgcatgcc aattatatct tctggcgtaa ttccacaatt atgactatgt cttacaatag 20400

tgtacttgat ttaagcaagt tcaattgtaa gcataaggct acagttgtca ttaatttaaa 20460

agattcatcc attagtgatg ttgtgttagg tttgttgaag aatggtaagt tgctagtgcg 20520

taataatgac gccatttgtg gtttttctaa tcatttggtc aacgtaaaca aatgaagtct 20580

ttaacctact tctggttgtt cttaccagta ctttcaacac ttagcctacc acaagatgtc 20640

accaggtgct cagctaacac taattttagg cggttctttt caaaatttaa tgttcaggcg 20700

cctgcagttg ttgtactggg cggttatcta cctattggtg aaaaccaggg tgtcaattca 20760

acttggtact gtgctggcca acatccaact gctagtggcg ttcatggtat ctttgttagc 20820

catattagag gtggtcatgg ctttgagatt ggcatttcgc aagagccttt tgaccctagt 20880

ggttaccagc tttatttaca taaggctact aacggtaaca ctaatgctac tgcgcgactg 20940

cgcatttgcc agtttcctag cattaaaaca ttgggcccca ctgctaataa tgatgttaca 21000

ataggtcgta attgcctatt taacaaagcc atcccagctc atatgagtga acatagtgtt 21060

gtcggcataa catgggataa tgatcgtgtc actgtctttt ctgacaagat ctattatttt 21120

tattttaaaa atgattggtc ccgtgttgcg acaaagtgtt acaacagtgg aggttgtgct 21180

atgcaatatg tttacgaacc cacctattac atgcttaatg ttactagtgc tggtgaggat 21240

ggtatttctt atcaaccctg tacagctaat tgcattggtt atgctgccaa tgtatttgct 21300

actgagccca atggccacat accagaaggt tttagtttta ataattggtt tcttttgtcc 21360

aatgattcca ctttggtgca tggtaaggtg gtttccaacc aaccattgtt ggtcaattgt 21420

cttttggcca ttcctaagat ttatggacta ggccaatttt tctcctttaa tcaaacgatc 21480

gatggtgttt gtaatggagc tgctgtgcag cgtgcaccag aggctctgag gtttaatatt 21540

aatgacacct ctgtcattct tgctgaaggc tcaattgtac ttcatactgc tttaggaaca 21600

aatttttctt ttgtttgcag taattcccca aatcctcact tagccacctt cgccatacct 21660

ctgggtgcta cccaagtacc ttattattgt tttcttaaag tggatactta caactccact 21720

gtttataaat ttttggctgt tttacctcct accgtcaggg aaattgtcat caccaagtat 21780

ggtgatgttt atgtcaatgg gtttggatac ttgcatctcg gtttgttgga tgctgtcaca 21840

attaatttca ctggtcatgg cactgacgat gatgtttctg gtttttggac catagcatcg 21900

actaattttg ttgatgcact catcgaagtt caaggaaccg ccattcagcg tattctttat 21960

tgtgatgatc ctgttagcca actcaagtgt tctcaggttg cttttgacct tgacgatggt 22020

ttttacccta tttcttctag aaaccttctg agtcatgaac agccaatttc ttttgttact 22080

ctgccatcat ttaatgatca ttcttttgtt aacattactg tatctgcttc ctttggtggt 22140

catagtggtg ccaaccttat tgcatctgac actactatca atgggtttag ttctttctgt 22200

gttgacacta gacaatttac catttcactg ttttataacg ttacaaacag ttatggttat 22260

gtgtctaaat cacaggacag taattgccct ttcaccttgc aatctgttaa tgattacctg 22320

tcttttagca aattttgtgt ttccaccagc cttttggcta gtgcctgtac catagatctt 22380

tttggttacc ctgagtttgg tagtggtgtt aagtttacgt ccctttactt tcaattcaca 22440

aagggtgagt tgattactgg cacgactaaa ccacttgaag gtgtcacgga cgtttctttt 22500

atgactctgg atgtgtgtac caagtatact atctatggct ttaaaggtga gggtatcatt 22560

acccttacaa attctagctt tttggcaggt gtttattaca catctgtttc tggacagttg 22620

ttagccttta agaatgtcac tagtggtgct gtttattctg ttacgccatg ttctttttca 22680

gagcaggctg catatgttga tgatgatata gtgggtgtta tttctagttt gtctagctcc 22740

acttttaaca gtactaggga gttgcctggt ttcttctacc attctaatga tggctctaat 22800

tgtacagagc ctgtgttggt gtatagtaac ataggtgttt gtaaatctgg cagtattggc 22860

tacgtcccat ctcagtctgg ccaagtcaag attgcaccca cggttactgg gaatattagt 22920

attcccacca actttagtat gagtattagg acagaatatt tacagcttta caacacgcct 22980

gttagtgttg attgtgccac atatgtttgt aatggtaact ctcgttgtaa acaattactc 23040

acccagtaca ctgcagcatg taagaccata gagtcagcat tacractcag cgctaggctt 23100

gagtctgttg aagttaactc tatgcttact atttctgaag aggctctaca gttagctacc 23160

attagttcgt ttaatggtga tggatataat tttactaatg tgctgggtgt ttctgtgtat 23220

gatcctgcaa ggggcagggt ggtacaaaaa aggtctttta ttgaagacct gctttttaat 23280

aaagtggtta ctaatggcct tggtactgtt gatgaagact ataagcgctg ttctaatggt 23340

cgctctgtgg cagatctagt ctgtgcacag tattactctg gtgtcatggt actacctggt 23400

gttgttgacg ctgagaagct tcacatgtat agtgcgtctc tcatcggtgg tatggtgcta 23460

ggaggtttta cttctgcagc ggcattgcct tttagctatg ctgttcaagc tagactcaat 23520

tatcttgctc tacagacgga tgttctacag cggaaccagc aattgcttgc tgagtctttt 23580

aactctgcta ttggtaatat aacttcagcc tttgagagtg ttaaagaggc tattagtcaa 23640

acttccaagg gtttgaacac tgtggctcat gcgcttacta aggttcaaga ggttgttaac 23700

tcgcagggtg cagctttgac tcaacttacc gtacagctgc aacacaactt ccaagccatt 23760

tctagttcta ttgatgacat ttactctcga ctggacattc tttcagccga tgttcaggtt 23820

gaccgtctca tcaccggcag attatcagca cttaatgctt ttgttgctca aaccctcact 23880

aagtatactg aggttcaggc tagcaggaag ttagcacagc aaaaggttaa tgagtgcgtt 23940

aaatcgcaat cccagcgtta tggtttttgt ggtggtgatg gcgagcacat tttctctctg 24000

gtacaggcag cacctcaggg cctgctgttt ttacatacag tacttgtacc gagtgatttt 24060

gtagatgtta ttgccatcgc tggcttatgc gttaacgatg aaattgcctt gactctacgt 24120

gagcctggct tagtcttgtt tacgcatgaa cttcaaaatc atactgcgac ggaatatttt 24180

gtttcatcgc gacgtatgtt tgaacctaga aaacctaccg ttagtgattt tgttcaaatt 24240

gagagttgtg tggtcaccta tgtcaatttg actagagacc aactaccaga tgtaatccca 24300

gattacatcg atgttaacaa aacacttgat gagattttag cttctctgcc caatagaact 24360

ggtccaagtc ttcctttaga tgtttttaat gccacttatc ttaatctcac tggtgaaatt 24420

gcagatttag agcagcgttc agagtctctc cgtaatacta cagaggagct ccaaagtctt 24480

atatataata tcaacaacac actagttgac cttgagtggc tcaaccgagt tgagacatat 24540

atcaagtggc cgtggtgggt ttggttgatt attttcattg ttctcatctt tgttgtgtca 24600

ttactagtgt tctgctgcat ttccacgggt tgttgtggat gctgcggctg ctgctgtgct 24660

tgtttctcag gttgttgtag gggtcctaga cttcaacctt acgaagtttt tgaaaaggtc 24720

cacgtgcagt gatgtttctt ggactttttc aatacacgat tgacacagtt gtcaaagatg 24780

tctcaaagtc tgctaacttg tctttggatg ctgtccaaga gttggagctc aatgtagttc 24840

caattagaca agcttcaaat gtgacgggtt ttcttttcac cagtgttttt atctacttct 24900

ttgcactgtt taaagcgtct tctttgaggc gcaattatat tatgttggca gcgcgttttg 24960

ctgtcattgt tctttattgc ccacttttat attattgtgg tgcattttta gatgcaacta 25020

ttatttgttg cacacttatt caaagtcggt ggcaggcttt gtttagtctg cttttactcc 25080

tggcgctata aaaatgcgct ctttattatt tttaatacta cgacactttc tttcctcaat 25140

ggtaaagcag ctttgacggc aaatccattg tgattttaga aggtggtgac cattacatca 25200

cttttggcaa ctcttttgtt gcttttgtta gtagcatcga cttgtatcta gctatacgtg 25260

ggcggcaaga agctgaccta cagctgttgc gaactgttga gcttcttgat ggcaagaagc 25320

tttatgtctt ttcgcaacat caaattgttg gcattactaa tgctgcattt gactcaattc 25380

aactagacga gtatgctaca attagtgaat gataatggtc tagtagttaa tgttatactt 25440

tggcttttcg tactcttttt cctgcttatt ataagcatta ctttcgtcca attggttaat 25500

ctgtgcttca cttgtcaccg gttgtgtaat agcgcagttt acacacctat agggcgtttg 25560

tatagagttt ataagtctta catgcaaata gaccccctcc ctagtactgt tattgacgta 25620

taaacgaaat atgtctaacg gttctattcc cgttgatgag gtgattcaac accttagaaa 25680

ctggaatttc acatggaata tcatactgac gatactactt gtagtgcttc agtatggcca 25740

ttacaagtac tctgcgttct tgtatggtgt caagatggct attctatgga tactttggcc 25800

tcttgtgtta gcactgtcac tttttgatgc atgggctagc tttcaggtca attgggtctt 25860

ttttgctttc agcatcctta tggcttgcat cactcttatg ctgtggataa tgtactttgt 25920

caatagcatt cggttgtggc gcaggacaca ttcttggtgg tctttcaatc ctgaaacaga 25980

cgcgcttctc actacttctg tgatgggccg acaggtctgc attccagtgc ttggagcacc 26040

aactggtgta acgctaacac tccttagtgg tacattgctt gtagagggct ataaggttgc 26100

tactggcgta caggtaagtc aattacctaa tttcgtcaca gtcgccaagg ccactacaac 26160

aattgtctac ggacgtgttg gtcgttcagt caatgcttca tctggcactg gttgggcttt 26220

ctatgtccgg tccaaacacg gcgactactc agctgtgagt aatccgagtt cggttctcac 26280

agatagtgag aaagtgcttc atttagtcta aacagaaact ttatggcttc tgtcagtttt 26340

caggatcgtg gccgcaaacg ggtgccatta tccctctatg cccctcttag ggttactaat 26400

gacaaacccc tttctaaggt acttgcaaat aatgctgtac ccactaataa aggaaataag 26460

gaccagcaaa ttggatactg gaatgagcaa attcgctggc gcatgcgccg tggtgagcga 26520

attgaacaac cttccaattg gcatttctac tacctcggaa caggacctca cgccgacctc 26580

cgctatagga ctcgtactga gggtgttttc tgggttgcta aagaaggcgc aaagactgaa 26640

cccactaacc tgggtgtcag aaaggcgtct gaaaagccaa ttattccaaa tttctctcaa 26700

cagcttccca gcgtagttga gattgttgaa cctaacacac ctcctacttc acgtgcaaat 26760

tcacgtagca ggagtcgtgg taatggcaac aacaggtcca gatctccaag taacaacaga 26820

ggcaataacc agtcccgcgg taattcacag aatcgtggaa ataaccaggg tcgtggagct 26880

tctcagaaca gaggaggcaa taataataac aataacaagt ctcgtaacca gtccaagaac 26940

agaaaccagt caaatgaccg tggtggtgta acatcacgcg atgatctggt ggctgctgtc 27000

aaggatgccc ttaaatcttt gggtattggc gaaaaccctg acaagcttaa gcaacagcag 27060

aagcccaaac aggaaaggtc tgacagcagc ggcaaaaata cacctaagaa gaacaaatcc 27120

agagccactt cgaaagaacg tgacctcaaa gacatcccag agtggaggag aattcccaag 27180

ggcgaaaata gcgtagcagc ttgcttcgga cccaggggag gcttcaaaaa ttttggagat 27240

gcggaatttg tcgaaaaagg tgttgatgcc tcaggctatg ctcagatcgc cagtttagca 27300

ccaaatgttg cagcattgct ctttggtggt aatgtggctg ttcgtgagct agcggactct 27360

tacgagatta catataatta taaaatgact gtgccaaagt ctgatccaaa tgtagagctt 27420

cttgtttcac aggtggatgc atttaaaact gggaatgcaa aaccccagag aaagaaggaa 27480

aagaagaaca agcgtgaaac cacgcagcag ctgaatgaag aggccatcta cgatgatgtg 27540

ggtgtgccat ctgatgtgac tcatgccaat ttggaatggg acacagctgt tgatggtggt 27600

gacacggccg ttgaaattat caacgagatc ttcgacacag gaaattaaac aatgtttgac 27660

tggcttatcc tggctatgtc ccagggtagt gccattacac tgttattact gagtgttttt 27720

ctagcgactt ggctgctggg ctatggcttt gccctctaac tagcggtctt ggtcttgcac 27780

acaacggtaa gccagtggta atgtcagtgc aagaaggata ttaccatagc actgtcatga 27840

ggggaacgca gtaccttttc atctaaacct ttgcacgagt aatcaaagat ccgcttgacg 27900

agcctatatg gaagagcgtg ccaggtattt gactcaagga ctgttagtaa ctgaagacct 27960

gacggtgttg atatgg 27976

<210> 34

<211> 4161

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV 1251-125-10 (125-10) passage30 spike protein CDS sequence

<400> 34

atgaagtctt taacctactt ctggttgttc ttaccagtac tttcaacact tagcctacca 60

caagatgtca ccaggtgctc agctaacact aattttaggc ggttcttttc aaaatttaat 120

gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga aaaccagggt 180

gtcaattcaa cttggtactg tgctggccaa catccaactg ctagtggcgt tcatggtatc 240

tttgttagcc atattagagg tggtcatggc tttgagattg gcatttcgca agagcctttt 300

gaccctagtg gttaccagct ttatttacat aaggctacta acggtaacac taatgctact 360

gcgcgactgc gcatttgcca gtttcctagc attaaaacat tgggccccac tgctaataat 420

gatgttacaa taggtcgtaa ttgcctattt aacaaagcca tcccagctca tatgagtgaa 480

catagtgttg tcggcataac atgggataat gatcgtgtca ctgtcttttc tgacaagatc 540

tattattttt attttaaaaa tgattggtcc cgtgttgcga caaagtgtta caacagtgga 600

ggttgtgcta tgcaatatgt ttacgaaccc acctattaca tgcttaatgt tactagtgct 660

ggtgaggatg gtatttctta tcaaccctgt acagctaatt gcattggtta tgctgccaat 720

gtatttgcta ctgagcccaa tggccacata ccagaaggtt ttagttttaa taattggttt 780

cttttgtcca atgattccac tttggtgcat ggtaaggtgg tttccaacca accattgttg 840

gtcaattgtc ttttggccat tcctaagatt tatggactag gccaattttt ctcctttaat 900

caaacgatcg atggtgtttg taatggagct gctgtgcagc gtgcaccaga ggctctgagg 960

tttaatatta atgacacctc tgtcattctt gctgaaggct caattgtact tcatactgct 1020

ttaggaacaa atttttcttt tgtttgcagt aattccccaa atcctcactt agccaccttc 1080

gccatacctc tgggtgctac ccaagtacct tattattgtt ttcttaaagt ggatacttac 1140

aactccactg tttataaatt tttggctgtt ttacctccta ccgtcaggga aattgtcatc 1200

accaagtatg gtgatgttta tgtcaatggg tttggatact tgcatctcgg tttgttggat 1260

gctgtcacaa ttaatttcac tggtcatggc actgacgatg atgtttctgg tttttggacc 1320

atagcatcga ctaattttgt tgatgcactc atcgaagttc aaggaaccgc cattcagcgt 1380

attctttatt gtgatgatcc tgttagccaa ctcaagtgtt ctcaggttgc ttttgacctt 1440

gacgatggtt tttaccctat ttcttctaga aaccttctga gtcatgaaca gccaatttct 1500

tttgttactc tgccatcatt taatgatcat tcttttgtta acattactgt atctgcttcc 1560

tttggtggtc atagtggtgc caaccttatt gcatctgaca ctactatcaa tgggtttagt 1620

tctttctgtg ttgacactag acaatttacc atttcactgt tttataacgt tacaaacagt 1680

tatggttatg tgtctaaatc acaggacagt aattgccctt tcaccttgca atctgttaat 1740

gattacctgt cttttagcaa attttgtgtt tccaccagcc ttttggctag tgcctgtacc 1800

atagatcttt ttggttaccc tgagtttggt agtggtgtta agtttacgtc cctttacttt 1860

caattcacaa agggtgagtt gattactggc acgactaaac cacttgaagg tgtcacggac 1920

gtttctttta tgactctgga tgtgtgtacc aagtatacta tctatggctt taaaggtgag 1980

ggtatcatta cccttacaaa ttctagcttt ttggcaggtg tttattacac atctgtttct 2040

ggacagttgt tagcctttaa gaatgtcact agtggtgctg tttattctgt tacgccatgt 2100

tctttttcag agcaggctgc atatgttgat gatgatatag tgggtgttat ttctagtttg 2160

tctagctcca cttttaacag tactagggag ttgcctggtt tcttctacca ttctaatgat 2220

ggctctaatt gtacagagcc tgtgttggtg tatagtaaca taggtgtttg taaatctggc 2280

agtattggct acgtcccatc tcagtctggc caagtcaaga ttgcacccac ggttactggg 2340

aatattagta ttcccaccaa ctttagtatg agtattagga cagaatattt acagctttac 2400

aacacgcctg ttagtgttga ttgtgccaca tatgtttgta atggtaactc tcgttgtaaa 2460

caattactca cccagtacac tgcagcatgt aagaccatag agtcagcatt acractcagc 2520

gctaggcttg agtctgttga agttaactct atgcttacta tttctgaaga ggctctacag 2580

ttagctacca ttagttcgtt taatggtgat ggatataatt ttactaatgt gctgggtgtt 2640

tctgtgtatg atcctgcaag gggcagggtg gtacaaaaaa ggtcttttat tgaagacctg 2700

ctttttaata aagtggttac taatggcctt ggtactgttg atgaagacta taagcgctgt 2760

tctaatggtc gctctgtggc agatctagtc tgtgcacagt attactctgg tgtcatggta 2820

ctacctggtg ttgttgacgc tgagaagctt cacatgtata gtgcgtctct catcggtggt 2880

atggtgctag gaggttttac ttctgcagcg gcattgcctt ttagctatgc tgttcaagct 2940

agactcaatt atcttgctct acagacggat gttctacagc ggaaccagca attgcttgct 3000

gagtctttta actctgctat tggtaatata acttcagcct ttgagagtgt taaagaggct 3060

attagtcaaa cttccaaggg tttgaacact gtggctcatg cgcttactaa ggttcaagag 3120

gttgttaact cgcagggtgc agctttgact caacttaccg tacagctgca acacaacttc 3180

caagccattt ctagttctat tgatgacatt tactctcgac tggacattct ttcagccgat 3240

gttcaggttg accgtctcat caccggcaga ttatcagcac ttaatgcttt tgttgctcaa 3300

accctcacta agtatactga ggttcaggct agcaggaagt tagcacagca aaaggttaat 3360

gagtgcgtta aatcgcaatc ccagcgttat ggtttttgtg gtggtgatgg cgagcacatt 3420

ttctctctgg tacaggcagc acctcagggc ctgctgtttt tacatacagt acttgtaccg 3480

agtgattttg tagatgttat tgccatcgct ggcttatgcg ttaacgatga aattgccttg 3540

actctacgtg agcctggctt agtcttgttt acgcatgaac ttcaaaatca tactgcgacg 3600

gaatattttg tttcatcgcg acgtatgttt gaacctagaa aacctaccgt tagtgatttt 3660

gttcaaattg agagttgtgt ggtcacctat gtcaatttga ctagagacca actaccagat 3720

gtaatcccag attacatcga tgttaacaaa acacttgatg agattttagc ttctctgccc 3780

aatagaactg gtccaagtct tcctttagat gtttttaatg ccacttatct taatctcact 3840

ggtgaaattg cagatttaga gcagcgttca gagtctctcc gtaatactac agaggagctc 3900

caaagtctta tatataatat caacaacaca ctagttgacc ttgagtggct caaccgagtt 3960

gagacatata tcaagtggcc gtggtgggtt tggttgatta ttttcattgt tctcatcttt 4020

gttgtgtcat tactagtgtt ctgctgcatt tccacgggtt gttgtggatg ctgcggctgc 4080

tgctgtgctt gtttctcagg ttgttgtagg ggtcctagac ttcaacctta cgaagttttt 4140

gaaaaggtcc acgtgcagtg a 4161

<210> 35

<211> 1386

<212> PRT

<213> porcine epidemic diarrhea virus

<220>

<221> misc_feature

<222> (838)..(838)

<223> Xaa can be any naturally occurring amino acid

<400> 35

Met Lys Ser Leu Thr Tyr Phe Trp Leu Phe Leu Pro Val Leu Ser Thr

1 5 10 15

Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe

20 25 30

Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val

35 40 45

Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr

50 55 60

Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly Ile

65 70 75 80

Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser

85 90 95

Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala

100 105 110

Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe

115 120 125

Pro Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr Ile

130 135 140

Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser Glu

145 150 155 160

His Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe

165 170 175

Ser Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg Val

180 185 190

Ala Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val Tyr

195 200 205

Glu Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly

210 215 220

Ile Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ala Ala Asn

225 230 235 240

Val Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser Phe

245 250 255

Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly Lys

260 265 270

Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro

275 280 285

Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp

290 295 300

Gly Val Cys Asn Gly Ala Ala Val Gln Arg Ala Pro Glu Ala Leu Arg

305 310 315 320

Phe Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val

325 330 335

Leu His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn Ser

340 345 350

Pro Asn Pro His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Thr Gln

355 360 365

Val Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val

370 375 380

Tyr Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile

385 390 395 400

Thr Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu

405 410 415

Gly Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp

420 425 430

Asp Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp

435 440 445

Ala Leu Ile Glu Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys

450 455 460

Asp Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu

465 470 475 480

Asp Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu

485 490 495

Gln Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe

500 505 510

Val Asn Ile Thr Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn

515 520 525

Leu Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val

530 535 540

Asp Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser

545 550 555 560

Tyr Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu

565 570 575

Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr

580 585 590

Ser Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu

595 600 605

Phe Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys

610 615 620

Gly Glu Leu Ile Thr Gly Thr Thr Lys Pro Leu Glu Gly Val Thr Asp

625 630 635 640

Val Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly

645 650 655

Phe Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala

660 665 670

Gly Val Tyr Tyr Thr Ser Val Ser Gly Gln Leu Leu Ala Phe Lys Asn

675 680 685

Val Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu

690 695 700

Gln Ala Ala Tyr Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu

705 710 715 720

Ser Ser Ser Thr Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr

725 730 735

His Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser

740 745 750

Asn Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln

755 760 765

Ser Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly Asn Ile Ser Ile

770 775 780

Pro Thr Asn Phe Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr

785 790 795 800

Asn Thr Pro Val Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn

805 810 815

Ser Arg Cys Lys Gln Leu Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr

820 825 830

Ile Glu Ser Ala Leu Xaa Leu Ser Ala Arg Leu Glu Ser Val Glu Val

835 840 845

Asn Ser Met Leu Thr Ile Ser Glu Glu Ala Leu Gln Leu Ala Thr Ile

850 855 860

Ser Ser Phe Asn Gly Asp Gly Tyr Asn Phe Thr Asn Val Leu Gly Val

865 870 875 880

Ser Val Tyr Asp Pro Ala Arg Gly Arg Val Val Gln Lys Arg Ser Phe

885 890 895

Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Asn Gly Leu Gly Thr

900 905 910

Val Asp Glu Asp Tyr Lys Arg Cys Ser Asn Gly Arg Ser Val Ala Asp

915 920 925

Leu Val Cys Ala Gln Tyr Tyr Ser Gly Val Met Val Leu Pro Gly Val

930 935 940

Val Asp Ala Glu Lys Leu His Met Tyr Ser Ala Ser Leu Ile Gly Gly

945 950 955 960

Met Val Leu Gly Gly Phe Thr Ser Ala Ala Ala Leu Pro Phe Ser Tyr

965 970 975

Ala Val Gln Ala Arg Leu Asn Tyr Leu Ala Leu Gln Thr Asp Val Leu

980 985 990

Gln Arg Asn Gln Gln Leu Leu Ala Glu Ser Phe Asn Ser Ala Ile Gly

995 1000 1005

Asn Ile Thr Ser Ala Phe Glu Ser Val Lys Glu Ala Ile Ser Gln

1010 1015 1020

Thr Ser Lys Gly Leu Asn Thr Val Ala His Ala Leu Thr Lys Val

1025 1030 1035

Gln Glu Val Val Asn Ser Gln Gly Ala Ala Leu Thr Gln Leu Thr

1040 1045 1050

Val Gln Leu Gln His Asn Phe Gln Ala Ile Ser Ser Ser Ile Asp

1055 1060 1065

Asp Ile Tyr Ser Arg Leu Asp Ile Leu Ser Ala Asp Val Gln Val

1070 1075 1080

Asp Arg Leu Ile Thr Gly Arg Leu Ser Ala Leu Asn Ala Phe Val

1085 1090 1095

Ala Gln Thr Leu Thr Lys Tyr Thr Glu Val Gln Ala Ser Arg Lys

1100 1105 1110

Leu Ala Gln Gln Lys Val Asn Glu Cys Val Lys Ser Gln Ser Gln

1115 1120 1125

Arg Tyr Gly Phe Cys Gly Gly Asp Gly Glu His Ile Phe Ser Leu

1130 1135 1140

Val Gln Ala Ala Pro Gln Gly Leu Leu Phe Leu His Thr Val Leu

1145 1150 1155

Val Pro Ser Asp Phe Val Asp Val Ile Ala Ile Ala Gly Leu Cys

1160 1165 1170

Val Asn Asp Glu Ile Ala Leu Thr Leu Arg Glu Pro Gly Leu Val

1175 1180 1185

Leu Phe Thr His Glu Leu Gln Asn His Thr Ala Thr Glu Tyr Phe

1190 1195 1200

Val Ser Ser Arg Arg Met Phe Glu Pro Arg Lys Pro Thr Val Ser

1205 1210 1215

Asp Phe Val Gln Ile Glu Ser Cys Val Val Thr Tyr Val Asn Leu

1220 1225 1230

Thr Arg Asp Gln Leu Pro Asp Val Ile Pro Asp Tyr Ile Asp Val

1235 1240 1245

Asn Lys Thr Leu Asp Glu Ile Leu Ala Ser Leu Pro Asn Arg Thr

1250 1255 1260

Gly Pro Ser Leu Pro Leu Asp Val Phe Asn Ala Thr Tyr Leu Asn

1265 1270 1275

Leu Thr Gly Glu Ile Ala Asp Leu Glu Gln Arg Ser Glu Ser Leu

1280 1285 1290

Arg Asn Thr Thr Glu Glu Leu Gln Ser Leu Ile Tyr Asn Ile Asn

1295 1300 1305

Asn Thr Leu Val Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr

1310 1315 1320

Ile Lys Trp Pro Trp Trp Val Trp Leu Ile Ile Phe Ile Val Leu

1325 1330 1335

Ile Phe Val Val Ser Leu Leu Val Phe Cys Cys Ile Ser Thr Gly

1340 1345 1350

Cys Cys Gly Cys Cys Gly Cys Cys Cys Ala Cys Phe Ser Gly Cys

1355 1360 1365

Cys Arg Gly Pro Arg Leu Gln Pro Tyr Glu Val Phe Glu Lys Val

1370 1375 1380

His Val Gln

1385

<210> 36

<211> 27976

<212> RNA

<213> Artificial Sequence

<220>

<223> PEDV 1251-125-10 (125-10) passage30 spike protein genomic RNA

<400> 36

aauucaacua aacgaaauuu uguccuuccg gccgcauguc caugcugcug gaagcugacg 60

uggaauuuca uuagguuugc uuaaguagcc aucgcaagug cugugcuguc cucuaguucc 120

ugguuggcgu uccgucgccu ucuacauacu agacaaacag ccuuccuccg guuccgucug 180

gggguugugu ggauaacuag uuccgucuag uuugaaacca guaacugucg gcuauggcua 240

gcaaccaugu uacauuggcu uuugccaaug augcagaaau uucagcuuuu ggcuuuugca 300

cugcuaguga agccgucuca uacuauucug aggccgccgc uaguggauuu augcaaugcc 360

guuucguguc cuucgaucuc gcugacacug uugagggauu gcuucccgaa gacuauguca 420

uggugguggu cggcacuacc aagcuuagug cguaugugga cacuuuuggu agccgcccca 480

aaaacauuug ugguuggcug uuauuuucua acuguaauua cuuccucgaa gaguuagagc 540

uuacuuuugg ucgucguggu gguaacaucg ugccaguuga ccaauacaug uguggcgcug 600

acgguaaacc uguucuucag gaauccgaau gggaguauac agauuucuuu gcugacuccg 660

aagacgguca acucaacauu gcugguauca cuuaugugaa ggccuggauu guagagcgau 720

cggaugucuc uuaugcgagu cagaauuuaa caucuauuaa gucuauuacu uacuguucaa 780

ccuaugagca uacuuuuccu gaugguacug ccaugaaggu ugcacguacu ccaaagauua 840

agaagacugu ugucuugucu gagccacuug cuacuaucua cagggaaauu gguucuccuu 900

uuguggauaa ugggagcgau gcucguucua ucauuaagag accaguguuc cuccacgcuu 960

uuguuaagug uaaguguggu aguuaucauu ggacuguugg ugauuggacu uccuaugucu 1020

ccacuugcug uggcuuuaag uguaagccag uccuuguggc uucaugcucu gcuacgccug 1080

guucuguugu gguuacgcgc gcuggugcug gcacuggugu uaaguauuac aacaacaugu 1140

uccugcgcca uguggcagac auugaugggu uggcauucug gcgaauucuc aaggugcagu 1200

ccaaagacga ccucgcuugc ucugguaaau uccuugaaca ccaugaggaa gguuucacag 1260

auccuugcua cuuuuugaau gacucgagca uugcuacuaa gcucaaguuu gacauccuua 1320

guggcaaguu uucugaugaa gucaaacaag cuaucuuugc uggucauguu guuguuggca 1380

gcgcgmucgu ugacauuguu gacgaugcac ugggacagcc uugguuuaua cguaagcuug 1440

gugaccuugc aagugcagcu ugggagcagc uuaaggcugu cguuagaggc cuuaaccucc 1500

ugucugauga ggucgugcuc uuuggcaaaa gacuuagcug ugccacucuu aguaucguua 1560

acgguguuuu ugaguucauc gccgaagugc cugagaaguu ggcugcggcu guuacaguuu 1620

uugucaacuu cuugaaugag cuuuuugagu cugccuguga cugcuuaaag gucggaggua 1680

aaaccuuuaa caagguuggc ucuuauguuc uuuuugacaa cgcauugguu aagcuuguca 1740

aggcaaaagu ucgcggccca cgacaggcag guguuuguga aguucguuac acaagccuug 1800

uuauugggag uacuaccaag gugguuucca agcgcguuga aaaugccaau gugaaucucg 1860

ucgucguuga cgaggaugug acccucaaca ccacuggucg uacaguuguu guugacggac 1920

uugcauucuu cgagagugac ggguuuuaca gacaucuugc ugaugcugac guugucauug 1980

aacauccugu uuauaagucu gcuugugagc ucaagccagu uuuugagugu gacccaauac 2040

cugauuuucc uaugccugug gccgcuagug uugcagagcu uugugugcaa acugaucugu 2100

ugcuuaaaaa uuacaacacu ccuuauaaaa cuuacagcug cguugugaga ggugauaagu 2160

guuguaucac uugcaccuua cauuucacag caccaaguua uauggaggcu gcugcuaauu 2220

uuguagaccu cuguaccaag aacauuggua cugcugguuu ucaugaguuu uacauuacgg 2280

cccaugaaca acaggaucug caaggguucg uaaccacuug uugcacgaug ucagguuuug 2340

aguguuuuau gccuauaauc ccacaguguc cagcagugcu ugaagagauu gaugguggua 2400

gcaucuggcg gucuuuuauc acuggucuua auacaaugug ggauuuuugc aagcaucuua 2460

aagucagcuu uggacuagau ggcauuguug ucacuguagc acgcaaauuu aaacgacuug 2520

gugcucucuu ggcagaaaug uauaacacuu accuuucaac ugugguggaa aacuugguac 2580

uggccggugu uagcuucaag uauuaugcca ccaguguccc aaaaauuguu uugggcuguu 2640

guuuucacag uguuaaaagu guucuugcaa gugccuucca gauuccuguc caggcaggcg 2700

uugagaaguu uaaagucuuc cuuaacugug uucacccugu uguaccacgu gucauugaaa 2760

cuucuuuugu ggaauuagaa gagacgacau uuaaaccacc agcacucaau gguaguauug 2820

cuauuguuga uggcuuugcu uucuauuaug auggaacacu auacuauccc accgauggua 2880

auagcguugu uccuaucugc uuuaagaaga aagguggugg ugaugucaaa uucucugaug 2940

aagucucugu uaaaaccauu gacccaguuu auaaggucuc ccuugaauuu gaguucgagu 3000

cugagacuau uauggcugug cuuaauaagg cuguugguaa uuguaucaag guuacaggug 3060

guugggacga uguuguugag uauaucaaug uugccauuga gguucuuaaa gaucacaucg 3120

augugccuaa guacuacauc uaugaugagg aagguggcac cgauccuaau cugcccguaa 3180

ugguuucuca guggccguug aaugaugaca cgaucucaca ggaucugcuu gauguugaag 3240

uuguuacuga ugcgccaguu gauuucgagg gugaugaagu agacuccucu gacccugwua 3300

agguggcaga cguggcuaac ucugagccug aggaugacgg ucuuaaugua gcuccugaaa 3360

caaauguaga gucugaaguu gaggaaguug ccgcaaccuu guccuuuaaa gauacaccuu 3420

ccacaguuac uaaggauccu uuugcuuuug acuuugcaag cuauggagga cuuaagguuu 3480

uaagacaauc ucauaacaac ugcuggguua cuucuaccuu ggugcagcua caauugcuug 3540

gcaucguuga ugacccugca auggagcuuu uuagugcugg uagaguuggu ccaaugguuc 3600

gcaaaugcua ugagucacaa aaggcuaucu ugggaucuuu gggugaugug ucggcuugcc 3660

uagagucucu gacuaaggac cuacacacac uuaagauuac cuguucugua gucugugguu 3720

gugguacugg ugaacguauc uaugaugguu gugcuuuucg uaugacgcca acuuuggaac 3780

cguucccaua uggugcuugu gcucagugug cucaaguuuu gaugcacacu uuuaaaagua 3840

uuguuggcac cggcaucuuu ugucgagaua cuacugcucu cuccuuggau ucuuugguug 3900

uaaaaccucu uugugcggcu gcuuuuauag gcaaggauag uggucauuau gucacuaacu 3960

uuuaugaugc ugcuauggcu auugaugguu auggucguca ucagauaaag uaugacacac 4020

ugaacacuau uuguguuaaa gacguuaauu ggacagcacc uuuuguccca gacguugagc 4080

cuguauugga gccuguuguc aaaccuuucu auucuuauaa gaauguugau uuuuaccaag 4140

gagauuuuag ugaccuuguu aaacuuccau gugauuuugu uguuaaugcu gcaaaugaga 4200

auuugucuca cgguggcggc auagcaaagg ccauugaugu uuauaccaag ggcauguugc 4260

agaagugcuc gaaugauuac auuaaagcac acggucccau uaaaguugga cgugguguca 4320

uguuggaggc auuaggucuu aaggucuuua auguuguugg uccacguaag gguaagcaug 4380

caccugagcu ucuuguuaag gcuuauaagu ccguuuuugc uaauucaggu guugcucuua 4440

caccuuugau uaguguugga auuuuuagug uuccuuugga agaaucuuua ucugcuuuuc 4500

uugcaugugu uggugaucgc cacuguaagu gcuuuuguua uagugacaaa gagcgcgagg 4560

cgaucauuaa uuacauggau ggcuugguag augcuauuuu caaagaugca cuuguugaua 4620

cuacuccugu ccaggaagau guucaacaag uuucacaaaa accaguuuug ccuaauuuug 4680

aaccuuucag gauugaaggu gcucaugcuu ucuaugagug caacccugaa gguuugaugu 4740

cauuaggugc ugacaagcug guguuguuua caaauuccaa uuuggauuuu uguagcguug 4800

guaagugucu uaacaaugug acuggcggug cauugcuuga agccauaaau guauuuaaaa 4860

agaguaacaa aacagugccu gcuggcaacu guguuacuuu ugagugugca gauaugauuu 4920

cuauuacuau gguaguauug ccaucugacg gugaugcuaa uuaugacaaa aauuaugcac 4980

gcgccgucgu caagguaucu aagcuuaaag gcaaguuauu gcuugcuguu ggugaugcca 5040

uguuguauuc caaguugucc caccucagcg uguuagguuu cguauccaca ccugaugaug 5100

uggagcguuu cuacgcaaau aagagugugg uuauuaaagu uacugaggau acacguagug 5160

uuaagacugu uaaaguagaa uccacuguua cuuauggaca acaaauugga ccuugucuug 5220

uuaaugacac cguugucaca gacaacaaac cuguuguugc ugauguugua gcuaagguug 5280

uaccaagugc uaauugggau ucacauuaug guuuugauaa ggcuggugag uuccacaugc 5340

uagaccauac uggguuugcc uuuccuagug aaguuguuaa cgguaggcgu gugcuuaaaa 5400

ccacagauaa uaacuguugg guuaauguua cauguuuaca auuacaguuu gcuagauuua 5460

gguucaaguc agcaggucua caggcuaugu gggaguccua uuguacuggu gauguugcua 5520

uguuugugca uugguuguac uggcuuacug guguugacaa aggucagccu agugauucag 5580

aaaaugcacu uaacauguug ucuaaguaca uuguuccugc ugguucuguc acuauugaac 5640

gugucacgca ugacgguugu uguuguagua agcguguugu cacugcacca guugugaaug 5700

cuagcguguu gaagcuuggc gucgaggaug gucuuugucc acauggucuu aacuacauug 5760

acaaaguugu uguaguuaaa gguacuacaa uuguugucaa uguuggaaaa ccuguagugg 5820

caccaucgca ccucuuucuu aaggguguuu ccuacacaac auuccuagau aaugguaacg 5880

guguugccgg ccauuauacu guuuuugauc augacacugg uauggugcau gauggagaug 5940

uuuuuguacc aggugaucuc aaugugucuc cuguuacaaa uguugucguc ucagagcaga 6000

cggcuguugu gauuaaagac ccugugaaga aaguagaguu agacgcuaca aagcuguuag 6060

acacuaugaa uuaugcaucg gaaagauucu uuuccuuugg ugauuuuaug ucacguaauu 6120

uaauuacagu guuuuuguac auccuuagua uuuugggucu cuguuuuagg gccuuucgua 6180

agagggaugu uaaaguucua gcugguguac cccaacguac ugguauuaua uugcguaaaa 6240

gugugcgcua uaaugcaaag gcuuugggug ucuucuucaa gcuaaaacuu uauugguuca 6300

aaguucuugg uaaguuuagu uuggguauuu augcauugua ugcauuacua uucaugacaa 6360

uacgcuuuac accuauaggu ggcccuguuu gugaugaugu uguugcuggu uaugcuaauu 6420

cuaguuuuga caagaaugag uauugcaaca guguuauuug uaaggucugu cucuaugggu 6480

accaggaacu uucggacuuc ucucacacac agguaguaug gcaacaccuu agagacccau 6540

uaauugguaa ugugaugccu uucuuuuauu uggcauuucu ggcaauuuuu ggggguguuu 6600

auguaaaggc uauuacucuc uauuuuauuu uccaguaucu uaacauacuu gguguguuuu 6660

ugggccuaca acaguccauu ugguuuuugc agcuugugcc uuuugauguc uuuggugacg 6720

agaucgucgu cuuuuucauc guuacacgcg uauugauguu ccuuaagcau guuuuccuug 6780

gcugcgauaa ggcaucuugu guggcuugcu cuaagagugc ucgccuuaag cgcguuccug 6840

uccagacuau uuuucagggu acuagcaaau ccuucuacgu acaugccaau ggugguucua 6900

aguucuguaa gaagcacaau uucuuuuguu uaaauuguga uucuuauggu ccaggcugca 6960

cuuuuauuaa ugacgucauu gcaacugaag uugguaaugu ugucaaacuu aaugugcaac 7020

cgacaggucc ugccacuauu cuuauugaca agguugaauu caguaauggu uuuuacuauc 7080

uuuauagugg ugacacauuu uggaaguaca acuuugacau aacagauaac aaauacacuu 7140

gcaaagaguc acuuaaaaau uguagcauaa ucacagacuu uauuguuuuu aacaauaaug 7200

guuccaaugu aaaucagguu aagaaugcau guguguauuu uucacagaug cuuuguaaac 7260

cuguuaaguu aguggacuca gcguuguugg ccaguuuguc uguugauuuu ggugcaagcu 7320

uacauagugc uuuuguuagu guguugucga auaguuuugg caaagaccug ucaaguugua 7380

augacaugca ggauugcaag agcacauugg guuuugauga uguaccauug gauaccuuua 7440

augcugcugu ugcugaggcu caucguuacg auguccucuu gacugacaug ucguucaaca 7500

auuuuaccac caguuaugca aaaccagagg aaaaacuucc cguccaugac auugccacgu 7560

guaugcgugu aggugccaag auuguuaauc auaacguucu ugucaaggau aguauaccug 7620

ugguguggcu uguacgugau uucauugccc uuucugaaga aacuaggaag uacauuauuc 7680

guacgacuaa aguuaagggu auaaccuuca uguugaccuu uaaugauugu cguaugcaua 7740

cuaccauacc uacuguuugc auugcaaaua agaagggugc aggucuuccu aguuuuucaa 7800

agguuaagaa auucuucugg uuuuuguguc uguucauagu ugcuguuuuc uuugcacuaa 7860

gcuuuuuuga uuuuaguacu cagguuagca gugauaguga uuaugacuuc aaguauauug 7920

agaguggcca guugaagacu uuugacaauc cacuuaguug ugugcauaau gucuuuagua 7980

acuucgacca guggcaugau gccaaguuug guuucacccc cgucaacaau ccuaguuguc 8040

cuauagucgu ugguguauca gacgaagcgc gcacuguucc agguauccca gcagguguuu 8100

auuuagcugg uaaaacacuu guuuuugcua uuaacaccau uuuugguaca ucugguuugu 8160

gcuuugaugc uaguggcguu gcugauaagg gcgcuugcau uuuuaauucg gcuugcacca 8220

cauuaucugg uuugggugga acugcugucu acuguuauaa gaauggucua guugaaggug 8280

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auuaauuuca cuggucaugg cacugacgau gauguuucug guuuuuggac cauagcaucg 21900

acuaauuuug uugaugcacu caucgaaguu caaggaaccg ccauucagcg uauucuuuau 21960

ugugaugauc cuguuagcca acucaagugu ucucagguug cuuuugaccu ugacgauggu 22020

uuuuacccua uuucuucuag aaaccuucug agucaugaac agccaauuuc uuuuguuacu 22080

cugccaucau uuaaugauca uucuuuuguu aacauuacug uaucugcuuc cuuugguggu 22140

cauaguggug ccaaccuuau ugcaucugac acuacuauca auggguuuag uucuuucugu 22200

guugacacua gacaauuuac cauuucacug uuuuauaacg uuacaaacag uuaugguuau 22260

gugucuaaau cacaggacag uaauugcccu uucaccuugc aaucuguuaa ugauuaccug 22320

ucuuuuagca aauuuugugu uuccaccagc cuuuuggcua gugccuguac cauagaucuu 22380

uuugguuacc cugaguuugg uagugguguu aaguuuacgu cccuuuacuu ucaauucaca 22440

aagggugagu ugauuacugg cacgacuaaa ccacuugaag gugucacgga cguuucuuuu 22500

augacucugg auguguguac caaguauacu aucuauggcu uuaaagguga ggguaucauu 22560

acccuuacaa auucuagcuu uuuggcaggu guuuauuaca caucuguuuc uggacaguug 22620

uuagccuuua agaaugucac uaguggugcu guuuauucug uuacgccaug uucuuuuuca 22680

gagcaggcug cauauguuga ugaugauaua guggguguua uuucuaguuu gucuagcucc 22740

acuuuuaaca guacuaggga guugccuggu uucuucuacc auucuaauga uggcucuaau 22800

uguacagagc cuguguuggu guauaguaac auagguguuu guaaaucugg caguauuggc 22860

uacgucccau cucagucugg ccaagucaag auugcaccca cgguuacugg gaauauuagu 22920

auucccacca acuuuaguau gaguauuagg acagaauauu uacagcuuua caacacgccu 22980

guuaguguug auugugccac auauguuugu aaugguaacu cucguuguaa acaauuacuc 23040

acccaguaca cugcagcaug uaagaccaua gagucagcau uacracucag cgcuaggcuu 23100

gagucuguug aaguuaacuc uaugcuuacu auuucugaag aggcucuaca guuagcuacc 23160

auuaguucgu uuaaugguga uggauauaau uuuacuaaug ugcugggugu uucuguguau 23220

gauccugcaa ggggcagggu gguacaaaaa aggucuuuua uugaagaccu gcuuuuuaau 23280

aaagugguua cuaauggccu ugguacuguu gaugaagacu auaagcgcug uucuaauggu 23340

cgcucugugg cagaucuagu cugugcacag uauuacucug gugucauggu acuaccuggu 23400

guuguugacg cugagaagcu ucacauguau agugcgucuc ucaucggugg uauggugcua 23460

ggagguuuua cuucugcagc ggcauugccu uuuagcuaug cuguucaagc uagacucaau 23520

uaucuugcuc uacagacgga uguucuacag cggaaccagc aauugcuugc ugagucuuuu 23580

aacucugcua uugguaauau aacuucagcc uuugagagug uuaaagaggc uauuagucaa 23640

acuuccaagg guuugaacac uguggcucau gcgcuuacua agguucaaga gguuguuaac 23700

ucgcagggug cagcuuugac ucaacuuacc guacagcugc aacacaacuu ccaagccauu 23760

ucuaguucua uugaugacau uuacucucga cuggacauuc uuucagccga uguucagguu 23820

gaccgucuca ucaccggcag auuaucagca cuuaaugcuu uuguugcuca aacccucacu 23880

aaguauacug agguucaggc uagcaggaag uuagcacagc aaaagguuaa ugagugcguu 23940

aaaucgcaau cccagcguua ugguuuuugu gguggugaug gcgagcacau uuucucucug 24000

guacaggcag caccucaggg ccugcuguuu uuacauacag uacuuguacc gagugauuuu 24060

guagauguua uugccaucgc uggcuuaugc guuaacgaug aaauugccuu gacucuacgu 24120

gagccuggcu uagucuuguu uacgcaugaa cuucaaaauc auacugcgac ggaauauuuu 24180

guuucaucgc gacguauguu ugaaccuaga aaaccuaccg uuagugauuu uguucaaauu 24240

gagaguugug uggucaccua ugucaauuug acuagagacc aacuaccaga uguaauccca 24300

gauuacaucg auguuaacaa aacacuugau gagauuuuag cuucucugcc caauagaacu 24360

gguccaaguc uuccuuuaga uguuuuuaau gccacuuauc uuaaucucac uggugaaauu 24420

gcagauuuag agcagcguuc agagucucuc cguaauacua cagaggagcu ccaaagucuu 24480

auauauaaua ucaacaacac acuaguugac cuugaguggc ucaaccgagu ugagacauau 24540

aucaaguggc cguggugggu uugguugauu auuuucauug uucucaucuu uguuguguca 24600

uuacuagugu ucugcugcau uuccacgggu uguuguggau gcugcggcug cugcugugcu 24660

uguuucucag guuguuguag ggguccuaga cuucaaccuu acgaaguuuu ugaaaagguc 24720

cacgugcagu gauguuucuu ggacuuuuuc aauacacgau ugacacaguu gucaaagaug 24780

ucucaaaguc ugcuaacuug ucuuuggaug cuguccaaga guuggagcuc aauguaguuc 24840

caauuagaca agcuucaaau gugacggguu uucuuuucac caguguuuuu aucuacuucu 24900

uugcacuguu uaaagcgucu ucuuugaggc gcaauuauau uauguuggca gcgcguuuug 24960

cugucauugu ucuuuauugc ccacuuuuau auuauugugg ugcauuuuua gaugcaacua 25020

uuauuuguug cacacuuauu caaagucggu ggcaggcuuu guuuagucug cuuuuacucc 25080

uggcgcuaua aaaaugcgcu cuuuauuauu uuuaauacua cgacacuuuc uuuccucaau 25140

gguaaagcag cuuugacggc aaauccauug ugauuuuaga agguggugac cauuacauca 25200

cuuuuggcaa cucuuuuguu gcuuuuguua guagcaucga cuuguaucua gcuauacgug 25260

ggcggcaaga agcugaccua cagcuguugc gaacuguuga gcuucuugau ggcaagaagc 25320

uuuaugucuu uucgcaacau caaauuguug gcauuacuaa ugcugcauuu gacucaauuc 25380

aacuagacga guaugcuaca auuagugaau gauaaugguc uaguaguuaa uguuauacuu 25440

uggcuuuucg uacucuuuuu ccugcuuauu auaagcauua cuuucgucca auugguuaau 25500

cugugcuuca cuugucaccg guuguguaau agcgcaguuu acacaccuau agggcguuug 25560

uauagaguuu auaagucuua caugcaaaua gacccccucc cuaguacugu uauugacgua 25620

uaaacgaaau augucuaacg guucuauucc cguugaugag gugauucaac accuuagaaa 25680

cuggaauuuc acauggaaua ucauacugac gauacuacuu guagugcuuc aguauggcca 25740

uuacaaguac ucugcguucu uguauggugu caagauggcu auucuaugga uacuuuggcc 25800

ucuuguguua gcacugucac uuuuugaugc augggcuagc uuucagguca auugggucuu 25860

uuuugcuuuc agcauccuua uggcuugcau cacucuuaug cuguggauaa uguacuuugu 25920

caauagcauu cgguuguggc gcaggacaca uucuuggugg ucuuucaauc cugaaacaga 25980

cgcgcuucuc acuacuucug ugaugggccg acaggucugc auuccagugc uuggagcacc 26040

aacuggugua acgcuaacac uccuuagugg uacauugcuu guagagggcu auaagguugc 26100

uacuggcgua cagguaaguc aauuaccuaa uuucgucaca gucgccaagg ccacuacaac 26160

aauugucuac ggacguguug gucguucagu caaugcuuca ucuggcacug guugggcuuu 26220

cuauguccgg uccaaacacg gcgacuacuc agcugugagu aauccgaguu cgguucucac 26280

agauagugag aaagugcuuc auuuagucua aacagaaacu uuauggcuuc ugucaguuuu 26340

caggaucgug gccgcaaacg ggugccauua ucccucuaug ccccucuuag gguuacuaau 26400

gacaaacccc uuucuaaggu acuugcaaau aaugcuguac ccacuaauaa aggaaauaag 26460

gaccagcaaa uuggauacug gaaugagcaa auucgcuggc gcaugcgccg uggugagcga 26520

auugaacaac cuuccaauug gcauuucuac uaccucggaa caggaccuca cgccgaccuc 26580

cgcuauagga cucguacuga ggguguuuuc uggguugcua aagaaggcgc aaagacugaa 26640

cccacuaacc ugggugucag aaaggcgucu gaaaagccaa uuauuccaaa uuucucucaa 26700

cagcuuccca gcguaguuga gauuguugaa ccuaacacac cuccuacuuc acgugcaaau 26760

ucacguagca ggagucgugg uaauggcaac aacaggucca gaucuccaag uaacaacaga 26820

ggcaauaacc agucccgcgg uaauucacag aaucguggaa auaaccaggg ucguggagcu 26880

ucucagaaca gaggaggcaa uaauaauaac aauaacaagu cucguaacca guccaagaac 26940

agaaaccagu caaaugaccg ugguggugua acaucacgcg augaucuggu ggcugcuguc 27000

aaggaugccc uuaaaucuuu ggguauuggc gaaaacccug acaagcuuaa gcaacagcag 27060

aagcccaaac aggaaagguc ugacagcagc ggcaaaaaua caccuaagaa gaacaaaucc 27120

agagccacuu cgaaagaacg ugaccucaaa gacaucccag aguggaggag aauucccaag 27180

ggcgaaaaua gcguagcagc uugcuucgga cccaggggag gcuucaaaaa uuuuggagau 27240

gcggaauuug ucgaaaaagg uguugaugcc ucaggcuaug cucagaucgc caguuuagca 27300

ccaaauguug cagcauugcu cuuugguggu aauguggcug uucgugagcu agcggacucu 27360

uacgagauua cauauaauua uaaaaugacu gugccaaagu cugauccaaa uguagagcuu 27420

cuuguuucac agguggaugc auuuaaaacu gggaaugcaa aaccccagag aaagaaggaa 27480

aagaagaaca agcgugaaac cacgcagcag cugaaugaag aggccaucua cgaugaugug 27540

ggugugccau cugaugugac ucaugccaau uuggaauggg acacagcugu ugaugguggu 27600

gacacggccg uugaaauuau caacgagauc uucgacacag gaaauuaaac aauguuugac 27660

uggcuuaucc uggcuauguc ccaggguagu gccauuacac uguuauuacu gaguguuuuu 27720

cuagcgacuu ggcugcuggg cuauggcuuu gcccucuaac uagcggucuu ggucuugcac 27780

acaacgguaa gccaguggua augucagugc aagaaggaua uuaccauagc acugucauga 27840

ggggaacgca guaccuuuuc aucuaaaccu uugcacgagu aaucaaagau ccgcuugacg 27900

agccuauaug gaagagcgug ccagguauuu gacucaagga cuguuaguaa cugaagaccu 27960

gacgguguug auaugg 27976

<210> 37

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> P1360110A PEDV 2a BD- FORWARD PRIMER N-terminal fragment

<400> 37

gatgtcacca ggtgctcagc taac 24

<210> 38

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV-S2-R PEDV 2a BD- REVERSE PRIMER N-terminal fragment

<400> 38

ctatacatgt gaagcttctc agcgtcaaca ac 32

<210> 39

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV-S1-F PEDV 2a BD- FORWARD PRIMER C-terminal fragment

<400> 39

catctgattc tggacagttg ttagcc 26

<210> 40

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> P1360110B.PEDV 2a BD- Reverse PRIMER C-terminal fragment

<400> 40

gaacactagt aatgacacaa caaagatgag aac 33

<210> 41

<211> 107

<212> DNA

<213> Artificial Sequence

<220>

<223> O1360110C gp64 signal sequence (AcNPV) cDNA

<400> 41

gatccgccac catggtatct gctattgttc tttatgtttt gcttgctgcc gccgctcact 60

cagctttcgc ggatgtcacc aggtgctcag ctaacactaa ttttagg 107

<210> 42

<211> 68

<212> DNA

<213> Artificial Sequence

<220>

<223> O1360110D gp64 C-terminal tail coding sequence (AcNPV) cDNA

<400> 42

gcttaaatac atatttagta ctgacgattg cggtttctga acactagtaa tgacacaaca 60

aagatgag 68

<210> 43

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194B Gp64 Forward Primer

<400> 43

gcttaaatac atatttagta c 21

<210> 44

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDV-S1-F Gp64 Reverse Primer

<400> 44

catctgattc tggacagttg ttagcc 26

<210> 45

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194A (OE Primer)

<400> 45

ggatccgcca ccatggtatc tg 22

<210> 46

<211> 4062

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDVS 2a BD Coding Sequence Expression Casette

<400> 46

atggtatctg ctattgttct ttatgttttg cttgctgccg ccgctcactc agctttcgcg 60

gatgtcacca ggtgctcagc taacactaat tttaggcggt tcttttcaaa atttaatgtt 120

caggcgcctg cagttgttgt actgggcggt tatctaccta ttggtgaaaa ccagggtgtc 180

aattcaactt ggtactgtgc tggccaacat ccaactgcta gtggcgttca tggtatcttt 240

gttagccata ttagaggtgg tcatggcttt gagattggca tttcgcaaga gccttttgac 300

cctagtggtt accagcttta tttacataag gctactaacg gtaacactaa tgctactgcg 360

cgactgcgca tttgccagtt tcctagcatt aaaacattgg gccccactgc taataatgat 420

gttacaacag gtcgtaattg cctatttaac aaagccatcc cagctcatat gagtgaacat 480

agtgttgtcg gcataacatg ggataatgat cgtgtcactg tcttttctga caagatctat 540

tatttttatt ttaaaaatga ttggtcccgt gttgcgacaa agtgttacaa cagtggaggt 600

tgtgctatgc aatatgttta cgaacccacc tattacatgc ttaatgttac tagtgctggt 660

gaggatggta tttcttatca accctgtaca gctaattgca ttggttatgc tgccaatgta 720

tttgctactg agcccaatgg ccacatacca gaaggtttta gttttaataa ttggtttctt 780

ttgtccaatg attccacttt ggtgcatggt aaggtggttt ccaaccaacc attgttggtc 840

aattgtcttt tggccattcc taagatttat ggactaggcc aatttttctc ctttaatcaa 900

acgatcgatg gtgtttgtaa tggagctgct gtgcagcgtg caccagaggc tctgaggttt 960

aatattaatg acacctctgt cattcttgct gaaggctcaa ttgtacttca tactgcttta 1020

ggaacaaatt tttcttttgt ttgcagtaat tcctcaaatc ctcacttagc caccttcgcc 1080

atacctttgg gtgctaccca agtaccctat tattgttttc ttaaagtgga tacttacaac 1140

tccactgttt ataaattctt ggctgtttta cctccaaccg tcagggaaat tgtcatcacc 1200

aagtatggtg atgtttatgt caatgggttt ggatacttgc atctcggttt gttggatgct 1260

gtcacaatta atttcactgg tcatggcact gacgatgatg tttctggttt ttggaccata 1320

gcatcgacta attttgttga tgcactcatc gaagttcaag gaaccgccat tcagcgtatt 1380

ctttattgtg atgatcctgt tagccaactc aagtgttctc aggttgcttt tgaccttgac 1440

gatggttttt accctatttc ttctagaaac cttctgagtc atgaacagcc aatttctttt 1500

gttactctgc catcatttaa tgatcattct tttgttaaca ttactgtatc tgcttccttt 1560

ggtggtcata gtggtgccaa ccttattgca tctgacacta ctatcaatgg gtttagttct 1620

ttctgtgttg acactagaca atttaccatt tcactgtttt ataacgttac aaacagttat 1680

ggttatgtgt ctaaatcaca ggacagtaat tgccctttca ccttgcaatc tgttaatgat 1740

tacctgtctt ttagcaaatt ttgtgtttcc accagccttt tggctagtgc ctgtaccata 1800

gatctttttg gttaccctga gtttggtagt ggtgttaagt ttacgtccct ttactttcaa 1860

ttcacaaagg gtgagttgat tactggcacg cctaaaccac ttgaaggtgt cacggacgtt 1920

tcttttatga ctctggatgt gtgtaccaag tatactatct atggctttaa aggtgagggt 1980

atcattaccc ttacaaattc tagctttttg gcaggtgttt attacacatc tgattctgga 2040

cagttgttag cctttaagaa tgtcactagt ggtgctgttt attctgttac gccatgttct 2100

ttttcagagc aggctgcata tgttgatgat gatatagtgg gtgttatttc tagtttgtct 2160

agctccactt ttaacagtac tagggagttg cctggtttct tctaccattc taatgatggc 2220

tctaattgta cagagcctgt gttggtgtat agtaacatag gtgtttgtaa atctggcagt 2280

attggctacg tcccatctca gtctggccaa gtcaagattg cacccacggt tactgggaat 2340

attagtattc ccaccaactt tagtatgagt attaggacag aatatttaca gctttacaac 2400

acgcctgtta gtgttgattg tgccacatat gtttgtaatg gtaactctcg ttgtaaacaa 2460

ttactcaccc agtacactgc agcatgtaag accatagagt cagcattaca actcagcgct 2520

aggcttgagt ctgttgaagt taactctatg cttactattt ctgaagaggc tctacagtta 2580

gctaccatta gttcgtttaa tggtgatgga tataatttta ctaatgtgct gggtgtttct 2640

gtgtatgatc ctgcaagtgg cagggtggta caaaaaaggt cttttattga agacctgctt 2700

tttaataaag tggttactaa tggccttggt actgttgatg aagactataa gcgctgttct 2760

aatggtcgct ctgtggcaga tctagtctgt gcacagtatt actctggtgt catggtacta 2820

cctggtgttg ttgacgctga gaagcttcac atgtatagtg cgtctctcat cggtggtatg 2880

gtgctaggag gttttacttc tgcagcggca ttgcctttta gctatgctgt tcaagctaga 2940

ctcaattatc ttgctctaca gacggatgtt ctacagcgga accagcaatt gcttgctgag 3000

tcttttaact ctgctattgg taatataact tcagcctttg agagtgttaa agaggctatt 3060

agtcaaactt ccaagggttt gaacactgtg gctcatgcgc ttactaaggt tcaagaggtt 3120

gttaactcgc agggtgcagc tttgactcaa cttaccgtac agctgcaaca caacttccaa 3180

gccatttcta gttctattga tgacatttac tctcgactgg acattctttc agccgatgtt 3240

caggttgacc gtctcatcac cggcagatta tcagcactta atgcttttgt tgctcaaacc 3300

ctcactaagt atactgaggt tcaggctagc aggaagttag cacagcaaaa ggttaatgag 3360

tgcgttaaat cgcaatctca gcgttatggt ttttgtggtg gtgatggcga gcacattttc 3420

tctctggtac aggcagcacc tcagggcctg ctgtttttac atacagtact tgtaccgagt 3480

gattttgtag atgttattgc catcgctggc ttatgcgtta acgatgaaat tgccttgact 3540

ctacgtgagc ctggcttagt cttgtttacg catgaacttc aaaatcatac tgcgacggaa 3600

tattttgttt catcgcgacg tatgtttgaa cctagaaaac ctaccgttag tgattttgtt 3660

caaattgaga gttgtgtggt cacctatgtc aatttgacta gagaccaact accagatgta 3720

atcccagatt acatcgatgt taacaaaaca cttgatgaga ttttagcttc tctgcccaat 3780

agaactggtc caagtcttcc tttagatgtt tttaatgcca cttatcttaa tctcactggt 3840

gaaattgcag atttagagca gcgttcagag tctctccgta atactacaga ggagctccaa 3900

agtcttatat ataatatcaa caacacacta gttgaccttg agtggctcaa ccgagttgag 3960

acatatatca agtggccgtg gtgggtttgg ttgattattt tcattgttct catctttgtt 4020

gtgtcattac tagtgttcag aaaccgcaat cgtcagtact aa 4062

<210> 47

<211> 1353

<212> PRT

<213> Artificial Sequence

<220>

<223> PEDVS 2a BD Amino Acid from Expression Casette

<400> 47

Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His

1 5 10 15

Ser Ala Phe Ala Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe Arg

20 25 30

Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val Leu

35 40 45

Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr Trp

50 55 60

Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly Ile Phe

65 70 75 80

Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser Gln

85 90 95

Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr

100 105 110

Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe Pro

115 120 125

Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr Thr Gly

130 135 140

Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser Glu His

145 150 155 160

Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ser

165 170 175

Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg Val Ala

180 185 190

Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val Tyr Glu

195 200 205

Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile

210 215 220

Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ala Ala Asn Val

225 230 235 240

Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser Phe Asn

245 250 255

Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly Lys Val

260 265 270

Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys

275 280 285

Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp Gly

290 295 300

Val Cys Asn Gly Ala Ala Val Gln Arg Ala Pro Glu Ala Leu Arg Phe

305 310 315 320

Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu

325 330 335

His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn Ser Ser

340 345 350

Asn Pro His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Thr Gln Val

355 360 365

Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr

370 375 380

Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr

385 390 395 400

Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly

405 410 415

Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp

420 425 430

Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp Ala

435 440 445

Leu Ile Glu Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys Asp

450 455 460

Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp

465 470 475 480

Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln

485 490 495

Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val

500 505 510

Asn Ile Thr Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu

515 520 525

Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp

530 535 540

Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr

545 550 555 560

Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln

565 570 575

Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser

580 585 590

Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe

595 600 605

Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly

610 615 620

Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val

625 630 635 640

Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe

645 650 655

Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala Gly

660 665 670

Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val

675 680 685

Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln

690 695 700

Ala Ala Tyr Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser

705 710 715 720

Ser Ser Thr Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr His

725 730 735

Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn

740 745 750

Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Ser

755 760 765

Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly Asn Ile Ser Ile Pro

770 775 780

Thr Asn Phe Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr Asn

785 790 795 800

Thr Pro Val Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser

805 810 815

Arg Cys Lys Gln Leu Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile

820 825 830

Glu Ser Ala Leu Gln Leu Ser Ala Arg Leu Glu Ser Val Glu Val Asn

835 840 845

Ser Met Leu Thr Ile Ser Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser

850 855 860

Ser Phe Asn Gly Asp Gly Tyr Asn Phe Thr Asn Val Leu Gly Val Ser

865 870 875 880

Val Tyr Asp Pro Ala Ser Gly Arg Val Val Gln Lys Arg Ser Phe Ile

885 890 895

Glu Asp Leu Leu Phe Asn Lys Val Val Thr Asn Gly Leu Gly Thr Val

900 905 910

Asp Glu Asp Tyr Lys Arg Cys Ser Asn Gly Arg Ser Val Ala Asp Leu

915 920 925

Val Cys Ala Gln Tyr Tyr Ser Gly Val Met Val Leu Pro Gly Val Val

930 935 940

Asp Ala Glu Lys Leu His Met Tyr Ser Ala Ser Leu Ile Gly Gly Met

945 950 955 960

Val Leu Gly Gly Phe Thr Ser Ala Ala Ala Leu Pro Phe Ser Tyr Ala

965 970 975

Val Gln Ala Arg Leu Asn Tyr Leu Ala Leu Gln Thr Asp Val Leu Gln

980 985 990

Arg Asn Gln Gln Leu Leu Ala Glu Ser Phe Asn Ser Ala Ile Gly Asn

995 1000 1005

Ile Thr Ser Ala Phe Glu Ser Val Lys Glu Ala Ile Ser Gln Thr

1010 1015 1020

Ser Lys Gly Leu Asn Thr Val Ala His Ala Leu Thr Lys Val Gln

1025 1030 1035

Glu Val Val Asn Ser Gln Gly Ala Ala Leu Thr Gln Leu Thr Val

1040 1045 1050

Gln Leu Gln His Asn Phe Gln Ala Ile Ser Ser Ser Ile Asp Asp

1055 1060 1065

Ile Tyr Ser Arg Leu Asp Ile Leu Ser Ala Asp Val Gln Val Asp

1070 1075 1080

Arg Leu Ile Thr Gly Arg Leu Ser Ala Leu Asn Ala Phe Val Ala

1085 1090 1095

Gln Thr Leu Thr Lys Tyr Thr Glu Val Gln Ala Ser Arg Lys Leu

1100 1105 1110

Ala Gln Gln Lys Val Asn Glu Cys Val Lys Ser Gln Ser Gln Arg

1115 1120 1125

Tyr Gly Phe Cys Gly Gly Asp Gly Glu His Ile Phe Ser Leu Val

1130 1135 1140

Gln Ala Ala Pro Gln Gly Leu Leu Phe Leu His Thr Val Leu Val

1145 1150 1155

Pro Ser Asp Phe Val Asp Val Ile Ala Ile Ala Gly Leu Cys Val

1160 1165 1170

Asn Asp Glu Ile Ala Leu Thr Leu Arg Glu Pro Gly Leu Val Leu

1175 1180 1185

Phe Thr His Glu Leu Gln Asn His Thr Ala Thr Glu Tyr Phe Val

1190 1195 1200

Ser Ser Arg Arg Met Phe Glu Pro Arg Lys Pro Thr Val Ser Asp

1205 1210 1215

Phe Val Gln Ile Glu Ser Cys Val Val Thr Tyr Val Asn Leu Thr

1220 1225 1230

Arg Asp Gln Leu Pro Asp Val Ile Pro Asp Tyr Ile Asp Val Asn

1235 1240 1245

Lys Thr Leu Asp Glu Ile Leu Ala Ser Leu Pro Asn Arg Thr Gly

1250 1255 1260

Pro Ser Leu Pro Leu Asp Val Phe Asn Ala Thr Tyr Leu Asn Leu

1265 1270 1275

Thr Gly Glu Ile Ala Asp Leu Glu Gln Arg Ser Glu Ser Leu Arg

1280 1285 1290

Asn Thr Thr Glu Glu Leu Gln Ser Leu Ile Tyr Asn Ile Asn Asn

1295 1300 1305

Thr Leu Val Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr Ile

1310 1315 1320

Lys Trp Pro Trp Trp Val Trp Leu Ile Ile Phe Ile Val Leu Ile

1325 1330 1335

Phe Val Val Ser Leu Leu Val Phe Arg Asn Arg Asn Arg Gln Tyr

1340 1345 1350

<210> 48

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194A PEDV 2b BD- FORWARD PRIMER N-terminal fragment

<400> 48

ggatccgcca ccatggtatc tg 22

<210> 49

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> P3183131B PEDV 2b BD- Reverse PRIMER N-terminal fragment

<400> 49

gtcgatgcta tggtccaaaa accag 25

<210> 50

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P3183131A PEDV 2b BD- FORWARD PRIMER C-terminal fragment

<400> 50

ggatccgcca ccatggtatc tg 22

<210> 51

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P290194B PEDV 2b BD- REVERSE PRIMER C-terminal fragment

<400> 51

gcttaaatac atatttagta c 21

<210> 52

<211> 4053

<212> DNA

<213> Artificial Sequence

<220>

<223> PEDVS 2b BD CDS cDNA

<400> 52

atggtatctg ctattgttct ttatgttttg cttgctgccg ccgctcactc agctttcgcg 60

gatgtcacta ggtgccagtc cactattaac ttcaggcggt tcttttcaaa atttaatgtg 120

caggcacctg ctgtcgttgt gttgggtggt tatctaccta gtatgaactc ctctagctgg 180

tactgtggca caggtcttga aactgctagt ggcgtgcatg gtattttcct cagttacatc 240

gattctggtc agggctttga gattggcatt tcacaggagc cgtttgatcc tagtggttac 300

cagctttatt tacataaggc cactaatggt aaccataatg ctattgcacg actgcgcatt 360

tgccagtttc caaataataa tacattgggc cctactgtta atgatgttac aacaggtcgt 420

aactgcctat tcaacaaagc cattccagct tatatgcagg atggaaaaaa catcgttgtc 480

ggcataacat gggacaatga tcgtgtcact gtttttgctg acaagatcta tcatttttat 540

ctcaaaaatg attggtcccg tgttgcgaca agatgttaca ataaaagaag ttgtgctatg 600

caatatgttt atacacctac ctactacatg cttaatgtta ctagtgcagg tgaggatggc 660

atttattatg aaccatgtac agctaattgc agtggttacg ctgccaatgt gtttgccact 720

gattctaatg gccacatacc agaaggtttt agttttaata attggtttct tttgtccaat 780

gattccactt tgttgcatgg taaggtggtt tccaaccaac ctttgttggt caattgtctt 840

ttggccattc ctaagattta tggactaggc caatttttct cattcaatca aacgatggat 900

ggcgtttgta atggagctgc tgcgcagcgt gcaccagagg ctctgaggtt taatattaat 960

gacacctctg tcattcttgc tgaaggctca attgtacttc acactgcttt aggaacaaat 1020

ctttcttttg tttgcagtaa ttcttcagat cctcatttag ctaccttcac catacctctg 1080

ggtgctaccc aagtacccta ttattgtttt cttaaagtgg atacttacaa ctccactgtt 1140

tataaatttt tggctgtttt acctcctacc gtcagggaaa ttgtcatcac caagtatggt 1200

gatgtttatg tcaatgggtt tggatacttg catctcggtt tgttggatgc tgtcacaatt 1260

aatttcactg gtcatggcac tgacgatgat gtttctggtt tttggaccat agcatcgact 1320

aattttgttg atgcactcat cgaagttcaa ggaaccgcca ttcagcgtat tctttattgt 1380

gatgatcctg ttagccaact caagtgttct caggttgctt ttgaccttga cgatggtttt 1440

taccctattt cttctagaaa ccttctgagt catgaacagc caatttcttt tgttactctg 1500

ccatcattta atgatcattc ttttgttaac attactgtat ctgcttcctt tggtggtcat 1560

agtggtgcca accttattgc atctgacact actatcaatg ggtttagttc tttctgtgtt 1620

gacactagac aatttaccat ttcactgttt tataacgtta caaacagtta tggttatgtg 1680

tctaaatcac aggacagtaa ttgccctttc accttgcaat ctgttaatga ttacctgtct 1740

tttagcaaat tttgtgtttc caccagcctt ttggctagtg cctgtaccat agatcttttt 1800

ggttaccctg agtttggtag tggtgttaag tttacgtccc tttactttca attcacaaag 1860

ggtgagttga ttactggcac gcctaaacca cttgaaggtg tcacggacgt ttcttttatg 1920

actctggatg tgtgtaccaa gtatactatc tatggcttta aaggtgaggg tatcattacc 1980

cttacaaatt ctagcttttt ggcaggtgtt tattacacat ctgattctgg acagttgtta 2040

gcctttaaga atgtcactag tggtgctgtt tattctgtta cgccatgttc tttttcagag 2100

caggctgcat atgttgatga tgatatagtg ggtgttattt ctagtttgtc tagctccact 2160

tttaacagta ctagggagtt gcctggtttc ttctaccatt ctaatgatgg ctctaattgt 2220

acagagcctg tgttggtgta tagtaacata ggtgtttgta aatctggcag tattggctac 2280

gtcccatctc agtctggcca agtcaagatt gcacccacgg ttactgggaa tattagtatt 2340

cccaccaact ttagtatgag tattaggaca gaatatttac agctttacaa cacgcctgtt 2400

agtgttgatt gtgccacata tgtttgtaat ggtaactctc gttgtaaaca attactcacc 2460

cagtacactg cagcatgtaa gaccatagag tcagcattac aactcagcgc taggcttgag 2520

tctgttgaag ttaactctat gcttactatt tctgaagagg ctctacagtt agctaccatt 2580

agttcgttta atggtgatgg atataatttt actaatgtgc tgggtgtttc tgtgtatgat 2640

cctgcaagtg gcagggtggt acaaaaaagg tcttttattg aagacctgct ttttaataaa 2700

gtggttacta atggccttgg tactgttgat gaagactata agcgctgttc taatggtcgc 2760

tctgtggcag atctagtctg tgcacagtat tactctggtg tcatggtact acctggtgtt 2820

gttgacgctg agaagcttca catgtatagt gcgtctctca tcggtggtat ggtgctagga 2880

ggttttactt ctgcagcggc attgcctttt agctatgctg ttcaagctag actcaattat 2940

cttgctctac agacggatgt tctacagcgg aaccagcaat tgcttgctga gtcttttaac 3000

tctgctattg gtaatataac ttcagccttt gagagtgtta aagaggctat tagtcaaact 3060

tccaagggtt tgaacactgt ggctcatgcg cttactaagg ttcaagaggt tgttaactcg 3120

cagggtgcag ctttgactca acttaccgta cagctgcaac acaacttcca agccatttct 3180

agttctattg atgacattta ctctcgactg gacattcttt cagccgatgt tcaggttgac 3240

cgtctcatca ccggcagatt atcagcactt aatgcttttg ttgctcaaac cctcactaag 3300

tatactgagg ttcaggctag caggaagtta gcacagcaaa aggttaatga gtgcgttaaa 3360

tcgcaatctc agcgttatgg tttttgtggt ggtgatggcg agcacatttt ctctctggta 3420

caggcagcac ctcagggcct gctgttttta catacagtac ttgtaccgag tgattttgta 3480

gatgttattg ccatcgctgg cttatgcgtt aacgatgaaa ttgccttgac tctacgtgag 3540

cctggcttag tcttgtttac gcatgaactt caaaatcata ctgcgacgga atattttgtt 3600

tcatcgcgac gtatgtttga acctagaaaa cctaccgtta gtgattttgt tcaaattgag 3660

agttgtgtgg tcacctatgt caatttgact agagaccaac taccagatgt aatcccagat 3720

tacatcgatg ttaacaaaac acttgatgag attttagctt ctctgcccaa tagaactggt 3780

ccaagtcttc ctttagatgt ttttaatgcc acttatctta atctcactgg tgaaattgca 3840

gatttagagc agcgttcaga gtctctccgt aatactacag aggagctcca aagtcttata 3900

tataatatca acaacacact agttgacctt gagtggctca accgagttga gacatatatc 3960

aagtggccgt ggtgggtttg gttgattatt ttcattgttc tcatctttgt tgtgtcatta 4020

ctagtgttca gaaaccgcaa tcgtcagtac taa 4053

<210> 53

<211> 1350

<212> PRT

<213> Artificial Sequence

<220>

<223> PEDVS 2b BD Expression Casetter Amino Acid Sequence

<400> 53

Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His

1 5 10 15

Ser Ala Phe Ala Asp Val Thr Arg Cys Gln Ser Thr Ile Asn Phe Arg

20 25 30

Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val Leu

35 40 45

Gly Gly Tyr Leu Pro Ser Met Asn Ser Ser Ser Trp Tyr Cys Gly Thr

50 55 60

Gly Leu Glu Thr Ala Ser Gly Val His Gly Ile Phe Leu Ser Tyr Ile

65 70 75 80

Asp Ser Gly Gln Gly Phe Glu Ile Gly Ile Ser Gln Glu Pro Phe Asp

85 90 95

Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr Asn Gly Asn His

100 105 110

Asn Ala Ile Ala Arg Leu Arg Ile Cys Gln Phe Pro Asn Asn Asn Thr

115 120 125

Leu Gly Pro Thr Val Asn Asp Val Thr Thr Gly Arg Asn Cys Leu Phe

130 135 140

Asn Lys Ala Ile Pro Ala Tyr Met Gln Asp Gly Lys Asn Ile Val Val

145 150 155 160

Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ala Asp Lys Ile

165 170 175

Tyr His Phe Tyr Leu Lys Asn Asp Trp Ser Arg Val Ala Thr Arg Cys

180 185 190

Tyr Asn Lys Arg Ser Cys Ala Met Gln Tyr Val Tyr Thr Pro Thr Tyr

195 200 205

Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile Tyr Tyr Glu

210 215 220

Pro Cys Thr Ala Asn Cys Ser Gly Tyr Ala Ala Asn Val Phe Ala Thr

225 230 235 240

Asp Ser Asn Gly His Ile Pro Glu Gly Phe Ser Phe Asn Asn Trp Phe

245 250 255

Leu Leu Ser Asn Asp Ser Thr Leu Leu His Gly Lys Val Val Ser Asn

260 265 270

Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys Ile Tyr Gly

275 280 285

Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Met Asp Gly Val Cys Asn

290 295 300

Gly Ala Ala Ala Gln Arg Ala Pro Glu Ala Leu Arg Phe Asn Ile Asn

305 310 315 320

Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu His Thr Ala

325 330 335

Leu Gly Thr Asn Leu Ser Phe Val Cys Ser Asn Ser Ser Asp Pro His

340 345 350

Leu Ala Thr Phe Thr Ile Pro Leu Gly Ala Thr Gln Val Pro Tyr Tyr

355 360 365

Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr Lys Phe Leu

370 375 380

Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr Lys Tyr Gly

385 390 395 400

Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly Leu Leu Asp

405 410 415

Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp Asp Val Ser

420 425 430

Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp Ala Leu Ile Glu

435 440 445

Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val

450 455 460

Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe

465 470 475 480

Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser

485 490 495

Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr

500 505 510

Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala Ser

515 520 525

Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg Gln

530 535 540

Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr Val

545 550 555 560

Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn

565 570 575

Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala

580 585 590

Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser Gly

595 600 605

Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile

610 615 620

Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met

625 630 635 640

Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe Lys Gly Glu

645 650 655

Gly Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala Gly Val Tyr Tyr

660 665 670

Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val Thr Ser Gly

675 680 685

Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln Ala Ala Tyr

690 695 700

Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser Ser Ser Thr

705 710 715 720

Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr His Ser Asn Asp

725 730 735

Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn Ile Gly Val

740 745 750

Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Ser Gly Gln Val

755 760 765

Lys Ile Ala Pro Thr Val Thr Gly Asn Ile Ser Ile Pro Thr Asn Phe

770 775 780

Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr Asn Thr Pro Val

785 790 795 800

Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser Arg Cys Lys

805 810 815

Gln Leu Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Ala

820 825 830

Leu Gln Leu Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu

835 840 845

Thr Ile Ser Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn

850 855 860

Gly Asp Gly Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp

865 870 875 880

Pro Ala Ser Gly Arg Val Val Gln Lys Arg Ser Phe Ile Glu Asp Leu

885 890 895

Leu Phe Asn Lys Val Val Thr Asn Gly Leu Gly Thr Val Asp Glu Asp

900 905 910

Tyr Lys Arg Cys Ser Asn Gly Arg Ser Val Ala Asp Leu Val Cys Ala

915 920 925

Gln Tyr Tyr Ser Gly Val Met Val Leu Pro Gly Val Val Asp Ala Glu

930 935 940

Lys Leu His Met Tyr Ser Ala Ser Leu Ile Gly Gly Met Val Leu Gly

945 950 955 960

Gly Phe Thr Ser Ala Ala Ala Leu Pro Phe Ser Tyr Ala Val Gln Ala

965 970 975

Arg Leu Asn Tyr Leu Ala Leu Gln Thr Asp Val Leu Gln Arg Asn Gln

980 985 990

Gln Leu Leu Ala Glu Ser Phe Asn Ser Ala Ile Gly Asn Ile Thr Ser

995 1000 1005

Ala Phe Glu Ser Val Lys Glu Ala Ile Ser Gln Thr Ser Lys Gly

1010 1015 1020

Leu Asn Thr Val Ala His Ala Leu Thr Lys Val Gln Glu Val Val

1025 1030 1035

Asn Ser Gln Gly Ala Ala Leu Thr Gln Leu Thr Val Gln Leu Gln

1040 1045 1050

His Asn Phe Gln Ala Ile Ser Ser Ser Ile Asp Asp Ile Tyr Ser

1055 1060 1065

Arg Leu Asp Ile Leu Ser Ala Asp Val Gln Val Asp Arg Leu Ile

1070 1075 1080

Thr Gly Arg Leu Ser Ala Leu Asn Ala Phe Val Ala Gln Thr Leu

1085 1090 1095

Thr Lys Tyr Thr Glu Val Gln Ala Ser Arg Lys Leu Ala Gln Gln

1100 1105 1110

Lys Val Asn Glu Cys Val Lys Ser Gln Ser Gln Arg Tyr Gly Phe

1115 1120 1125

Cys Gly Gly Asp Gly Glu His Ile Phe Ser Leu Val Gln Ala Ala

1130 1135 1140

Pro Gln Gly Leu Leu Phe Leu His Thr Val Leu Val Pro Ser Asp

1145 1150 1155

Phe Val Asp Val Ile Ala Ile Ala Gly Leu Cys Val Asn Asp Glu

1160 1165 1170

Ile Ala Leu Thr Leu Arg Glu Pro Gly Leu Val Leu Phe Thr His

1175 1180 1185

Glu Leu Gln Asn His Thr Ala Thr Glu Tyr Phe Val Ser Ser Arg

1190 1195 1200

Arg Met Phe Glu Pro Arg Lys Pro Thr Val Ser Asp Phe Val Gln

1205 1210 1215

Ile Glu Ser Cys Val Val Thr Tyr Val Asn Leu Thr Arg Asp Gln

1220 1225 1230

Leu Pro Asp Val Ile Pro Asp Tyr Ile Asp Val Asn Lys Thr Leu

1235 1240 1245

Asp Glu Ile Leu Ala Ser Leu Pro Asn Arg Thr Gly Pro Ser Leu

1250 1255 1260

Pro Leu Asp Val Phe Asn Ala Thr Tyr Leu Asn Leu Thr Gly Glu

1265 1270 1275

Ile Ala Asp Leu Glu Gln Arg Ser Glu Ser Leu Arg Asn Thr Thr

1280 1285 1290

Glu Glu Leu Gln Ser Leu Ile Tyr Asn Ile Asn Asn Thr Leu Val

1295 1300 1305

Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr Ile Lys Trp Pro

1310 1315 1320

Trp Trp Val Trp Leu Ile Ile Phe Ile Val Leu Ile Phe Val Val

1325 1330 1335

Ser Leu Leu Val Phe Arg Asn Arg Asn Arg Gln Tyr

1340 1345 1350

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