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1. 发明申请

WO2010135054A3 MUTATIONS IN A TOLL-LIKE RECEPTOR MOTIF IN THE NS4B OF CLASSICAL SWINE FEVER VIRUS SKIN BRESCIA INFLUENCES VIRULENCE IN SWINE 审中-公开
标题翻译：经典蛇类病毒NS4B类似感受态细胞中的突变体病毒感染西班牙血症
公开(公告)号：WO2010135054A3
公开(公告)日：2011-02-17
申请号：PCT/US2010032151
申请日：2010-04-23
申请人： US AGRICULTURE , BORCA MANUEL V , ZHU JAMES J
发明人： BORCA MANUEL V , ZHU JAMES J
IPC分类号： C12N15/40 , A61K39/12 , C07K14/08 , C12N7/01 , C12N15/63
CPC分类号： A61K39/12 , A61K2039/5254 , A61K2039/543 , A61K2039/552 , C07K14/005 , C12N7/00 , C12N2770/24322 , C12N2770/24334 , C12N2770/24362
摘要： NS4B is one of the non-structural proteins of classical swine fever virus. By using functional genetics, we have discovered, in the predicted amino acid sequence of NS4B of CSFV strain Brescia, a motif that resembles those found in the toll-like receptor (TLR) proteins, a group of host cell proteins involved in the development of anti-viral mechanisms. We have located the TLR motif in two groups of amino acid triplets at amino acid positions 2531 -3 (residues IYK) and 2566-8 (residues VGI) of the CSFV NS4B glycoprotein. We have constructed a recombinant CSFV (derived from an infectious clone containing the genetic information of the highly virulent strain Brescia) containing amino acid substitutions in the three amino acid residues at positions 2566, 2567 and 2568, where the VGI triplet has been replaced by an AAA triplet inside the NS4B glycoprotein. The obtained virus, named NS4B-VGIv, was completely attenuated in swine, showing a limited ability in spreading during the infection in vivo. Although attenuated, NS4B-VGIv efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection.
摘要翻译： NS4B是经典猪瘟病毒的非结构蛋白之一。通过使用功能遗传学，我们已经发现，在CSFV菌株布雷西亚的NS4B的预测氨基酸序列中，类似于在toll样受体（TLR）蛋白中发现的基序，一组参与发展的宿主细胞蛋白抗病毒机制。我们将TLR基序定位在CSFV NS4B糖蛋白的氨基酸位置2531-3（残基IYK）和2566-8（残基VGI）的两组氨基酸三联体中。我们已经构建了重组CSFV（来自含有高毒力菌株Brescia的遗传信息的感染性克隆），其含有在位置2566,2567和2568处的三个氨基酸残基中的氨基酸取代，其中VGI三联体被 NS4B糖蛋白内的AAA三联体。获得的病毒NS4B-VGIv在猪中完全衰减，在体内感染期间展开的能力有限。尽管减毒，NS4B-VGIv在感染后3和28天有效地保护猪免受有毒BICv的攻击。

2. 发明申请

WO2010047955A3 N-linked glycosylation alteration in e0 and e2 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine 审中-公开
标题翻译：蚀变糖基化N联糖蛋白E0和E2病毒猪瘟旧版和新版病毒疫苗猪瘟CLASSIC
公开(公告)号：WO2010047955A3
公开(公告)日：2010-07-08
申请号：PCT/US2009059920
申请日：2009-10-08
申请人： US AGRICULTURE , BORCA MANUEL V , RISATTI GUILLERMO R
发明人： BORCA MANUEL V , RISATTI GUILLERMO R
IPC分类号： C12N15/33 , A61K39/12 , C12N7/00
CPC分类号： G01N33/56983 , A61K39/12 , A61K39/187 , A61K2039/5254 , A61K2039/543 , A61K2039/552 , C07K14/005 , C12N7/00 , C12N2770/24322 , C12N2770/24334 , C12N2770/24362 , C12Q1/701 , G01N2333/183
摘要： E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E2 is involved in several functions including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Seven putative glycosylation sites in E2 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2, but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N1 16 (N 1 v virus) was responsible for BICv attenuation. N1 v efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection suggesting that glycosylation of E2 could be modified for development of CSF live-attenuated vaccines. Additionally, a new developed virus, contained deletions of putative glycosylation sites N1 in E2 and N 1 in EO (6b), called N1 E0/2v, induce a solid protection against the challenge at 3 and 28 days post-inoculation.
摘要翻译： E2是猪瘟（CSFV）的病毒的三个包膜糖蛋白之一。 E2参与多种功能，包括结合和进入病毒的靶细胞，抗体产生，在猪的保护性免疫应答的诱导，和毒力。在E2九月假定基化位点被CSFV布雷西亚（BICv）的感染性克隆的定点诱变来修饰。获得病毒突变体面板和用于研究在E2糖公认的糖基化位点的消除是否会影响猪的毒力/病毒致病。我们发现，可行的拯救病毒完全被去除E2所有公认的糖基化位点的改变，但恢复时在野生型天冬酰胺恢复N185A突变产生这是在猪减毒活病毒。每个E2糖基化位点的点突变证明，16 L1氨基酸（N1v病毒）负责BICv的衰减。 N1v有效保护猪抵抗暴露于致命的BICv 3和28天后感染，暗示E2糖基化可以为对抗CSF减毒活疫苗的研制进行修改。另外，含有E2和N1缺失N1假定基化位点在E0（6B）新开发的病毒，称为N1 E0 / 2体积诱导针对曝光一个牢固的保护，以3和28天接种后。

3. 发明申请

WO2009091720A3 N-linked glycosylation alteration in e1 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine 审中-公开
标题翻译：的糖基化N IN CONNECTION糖蛋白E1 VIRUS猪瘟CLASSIC蚀变和疫苗对病毒
公开(公告)号：WO2009091720A3
公开(公告)日：2009-10-08
申请号：PCT/US2009030824
申请日：2009-01-13
申请人： US AGRICULTURE , BORCA MANUEL V , RISATTI GUILLERMO R
发明人： BORCA MANUEL V , RISATTI GUILLERMO R
IPC分类号： C12N15/33
CPC分类号： C07H21/04 , A61K39/12 , A61K2039/5254 , A61K2039/552 , C12N7/00 , C12N2770/24334 , C12N2770/24362 , G01N33/56983 , G01N2333/183
摘要： E1, along with Ems and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Our previous studies indicated that glycosylation status of either E2 or Erns strongly influence viral virulence in swine. Here, we have investigated the role of E1 glycosylation of highly virulent CSFV strain Brescia during infection in the natural host. The three putative glycosylation sites in E1 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E1 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all three putative glycosylation sites in E1. Single mutations of each of the E1 glycosylation sites showed that CSFV amino acid N594 (E1.N3 virus), as well the combined mutation of N500 and N513 (E1.N1N2 virus) resulted in BICv attenuation. Infection of either E1. N1N2 or E1.N3 viruses were able to efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. These results, along with those demonstrating the role of glycosylation of Erps and E2, suggest that manipulation of the pattern of glycosylation could be a useful tool for development of CSF live-attenuated vaccines.
摘要翻译：根据本发明，E1，和E2用EMS，猪瘟（CSFV）的病毒的三个包膜糖蛋白中的一个。我们以往的研究表明，糖基化状态E2或Erns的强烈影响猪的病毒毒力。本发明是对病毒的高毒力株布雷西亚的E1糖基化的天然宿主的感染过程中的作用的研究结果。在E1的三个假定基化位点被CSFV布雷西亚（BICv）的感染性克隆的定点诱变来修饰。得到的一组突变体病毒的并用于确定在E1糖蛋白中除去推定的糖基化位点的改变是否在猪的毒力/病毒致病。据观察，在可行的拯救病毒完全被去除的E1三个假定基化位点改变。每个E1糖基化位N594的单突变表明CSFV氨基酸（E1.N3病毒），和N500和N513（E1.N1N2病毒）的组合突变导致衰减BICv。感染病毒或E1.N1N2 E1.N3，有效防止有毒BICv，3和感染28天后的猪。这些结果，与糖基化的Erns和E2的作用结果相结合表明，糖基化模式的操作是在减弱的猪瘟活疫苗发展的有用工具。

4. 发明申请

WO2007143442A3 A NOVEL VIRULENCE DETERMINANT WITHIN THE E2 STRUCTURAL GLYCOPROTEIN OF CLASSICAL SWINE FEVER VIRUS 审中-公开
标题翻译：经典蛇型病毒的E2结构型GLYCOPROTEIN中的新型毒力测定
公开(公告)号：WO2007143442A3
公开(公告)日：2008-03-13
申请号：PCT/US2007069852
申请日：2007-05-29
申请人： US AGRICULTURE , BORCA MANUEL V , RISATTI GUILLERMO R
发明人： BORCA MANUEL V , RISATTI GUILLERMO R
IPC分类号： A61K39/12 , A61K39/00 , C12N15/09 , C12Q1/68
CPC分类号： A61K39/12 , A61K2039/5254 , C07K14/005 , C12N7/00 , C12N2770/24322 , C12N2770/24334 , C12N2770/24361
摘要： Classical Swine Fever Virus (CSFV) E2 glycoprotein is a major inducer of neutralizing antibodies and protective immunity in swine. E2 mediates virus adsorption to the target cell, and harbors genetic determinants associated with virus virulence. CSFV E2 also contains between residues 829 and 837 a discrete epitope (TAVSPTTLR) recognized by monoclonal antibody (mAb) WH3O3, used to differentiate CSFV from related Pestiviruses Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV). In this report, a CSFV infectious clone of the virulent Brescia isolate (BICv) was used to progressively mutate the mAb WH3O3 epitope of CSFV E2 to the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). While the resulting virus mutants TIv (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) demonstrated in vitro growth characteristics similar to those of parental BICv, mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and a significant decrease in plaque size relative to parental BICv. Immunohistochemical reactivity with WH303 was lost only in T3v, T4v and T5v.
摘要翻译：经典猪瘟病毒（CSFV）E2糖蛋白是猪的中和抗体和保护性免疫的主要诱导剂。 E2介导病毒对靶细胞的吸附，并且具有与病毒毒力相关的遗传决定簇。 CSFV E2还包含残基829和837之间由单克隆抗体（mAb）WH3O3识别的离散表位（TAVSPTTLR），用于区分来自相关的病毒性腹泻病毒（BVDV）和边界病病毒（BDV）的CSFV。在本报告中，使用强毒性布雷西亚分离株（BICv）的CSFV感染性克隆逐渐将CSFV E2的mAb WH3O3表位突变为BVDV毒株NADL E2（TSFNMDTLA）的同源氨基酸序列。尽管得到的病毒突变体TIv（TSFSPTTLR），T2v（TSFNPTTLR），T3v（TSFNMTLTLR）证明体外生长特征与亲代BICv相似，但突变体T4v（TSFNMDTLR）和T5v（TSFNMDTLA）的病毒产量降低了10倍相对于亲本BICv，斑块大小明显减少。仅在T3v，T4v和T5v中，与WH303的免疫组织化学反应丧失。

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