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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amino acid substitutions in the three amino acid residues at positions 2566, 2567 and 2568, where the VGI triplet has been replaced by an AAA triplet inside the NS4B glycoprotein.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

WO2007143442A2 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：WO2007143442A2
公开(公告)日：2007-12-13
申请号：PCT/US2007/069852
申请日：2007-05-29
申请人： THE UNITED STATES OF AMERICA, AS REPRESNTED BY THE SECRETARY OF AGRICULTURE , BORCA, Manuel, V. , RISATTI, Guillermo, R.
发明人： BORCA, Manuel, V. , RISATTI, Guillermo, R.
IPC分类号： A61K39/12 , C12Q1/70 , C12N7/00
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) WH303, 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 WH303 epitope of CSFV E2 to the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). While the resulting virus mutants T1v (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. lmmunohistochemical reactivity with WH303 was lost only in T3v, T4v and T5v. Interestingly, progressive mutation of the WH303 epitope had an additive effect on attenuation for the virus in swine, with mutants T1v, T2v or T3v inducing progressively milder but invariably lethal CSF, T4v inducing only mild and transient clinical disease, and T5v inducing no disease. Swine infected with either T4v or T5v showed decreased virus replication in tonsils, draining lymph node, spleen and kidney and a significant reduction in virus shedding. Finally, T5v-infected animals were protected from clinical disease when challenged with virulent Brescia virus at 3 or 21 days post T5v inoculation. These results indicate that amino acid residues 830 to 834 of E2 are critical for virulence of CSFV in swine and that engineering at this locus may provide basis for a rationally designed live attenuated CSF vaccine.
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）WH303识别的离散表位（TAVSPTTLR），用于区分CSFV与相关的Pestiviruses牛病毒性腹泻病毒（BVDV）和边界病病毒（BDV）的区别。在本报告中，使用强毒性布雷西亚分离株（BICv）的CSFV感染性克隆将CSFV E2的mAb WH303表位逐渐突变为BVDV毒株NADL E2（TSFNMDTLA）的同源氨基酸序列。虽然得到的病毒突变体T1v（TSFSPTTLR），T2v（TSFNPTTLR），T3v（TSFNMTTLR）表现出与亲本BICv相似的体外生长特征，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。有趣的是，WH303表位的进行性突变对猪的病毒的衰减具有加性作用，突变体T1v，T2v或T3v诱导逐渐温和但始终致命的CSF，T4v仅诱导轻度和短暂的临床疾病，T5v诱导无疾病。感染T4v或T5v的猪显示扁桃体病毒复制减少，引流淋巴结，脾脏和肾脏，病毒脱落明显减少。最后，T5v感染的动物在T5v接种3天或21天时受到毒力布列西亚病毒攻击时被保护免于临床疾病。这些结果表明E2的氨基酸残基830至834对于猪中CSFV的毒力是至关重要的，并且在该基因座的工程可为合理设计的减毒活疫苗疫苗提供依据。

4. 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。

5. 发明申请

WO2008147799A1 A LIVE ATTENUATED ANTIGENICALLY MARKED CLASSICAL SWINE FEVER VACCINE 审中-公开
标题翻译：一个有力的抗衰老标签的经典的SWINE FEER VACCINE
公开(公告)号：WO2008147799A1
公开(公告)日：2008-12-04
申请号：PCT/US2008/064322
申请日：2008-05-21
申请人： THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF AGRICULTURE , BORCA, Manuel, V. , RISATTI, Guillermo, R.
发明人： BORCA, Manuel, V. , RISATTI, Guillermo, R.
IPC分类号： A61K39/12 , A61K39/00
CPC分类号： C12N7/00 , A61K2039/5254 , C07K14/005 , C07K2319/00 , C07K2319/43 , C12N2770/24322 , C12N2770/24361
摘要： Classical swine fever virus is a world-wide distributed highly contagious disease affecting swine. The two main strategies for diseases control are prophylactic vaccination and non-vaccination stamping out policies. Marker vaccines are a promising strategy. Here we report the rational development of a doubly antigenic marker CSFV experimental live attenuated candidate strain vaccine (Flag/T4 virus). Flag/T virus (Flag/T4v) is based in the combination of two Brescia derived recombinant attenuated viruses: RB-C22 and T4. RB-C22v contains a 19mer insertion in the structural glycoprotein E1, while T4v posses mutated CSFV amino acid residues 830 to 834 in the structural glycoprotein E2, deleting the highly conserved epitope recognized by monoclonal antibody (mAb) WH303. Flag/T4 virus contains a positive foreign antigenic marker, due to the insertion of the highly antigenic epitope Flag in the 19mer insertion of E1, as well as a negative antigenic marker, the lack of reactivity with mAb WH303. Immunized with Flag/T4v induced a complete protection against the challenge with virulent strain Brescia both at 3 and 28 days post infection when nasally administered and since the second day post infection when intramuscularly administered. These results constitute an example of rational design of a CSFV antigenically marked LAV.
摘要翻译：古典猪瘟病毒是世界范围内分布极高的传染性疾病，影响猪。疾病控制的两个主要策略是预防性接种疫苗和非疫苗接种策略。标记疫苗是一个有希望的策略。在这里我们报告双重抗原标志物CSFV实验性减毒候选菌株疫苗（Flag / T4病毒）的合理开发。 Flag / T病毒（Flag / T4v）基于两种Brescia衍生的重组减毒病毒：RB-C22和T4的组合。 RB-C22v在结构糖蛋白E1中包含19mer插入，而T4v在结构性糖蛋白E2中具有突变的CSFV氨基酸残基830至834，从而缺失由单克隆抗体（mAb）WH303识别的高度保守的表位。 Flag / T4病毒含有阳性外源性抗原标志物，因为插入E1抗原表位的高度抗原标记，以及阴性抗原标记，与mAb WH303缺乏反应性。用Flag / T4v免疫后，在经鼻给药后3天和28天以及肌内注射后感染后第二天，均能诱导出抗毒株Brescia的完整保护。这些结果构成了CSFV抗原标记LAV的合理设计的一个例子。

6. 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。

7. 发明申请

WO2010135054A2 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中类似感受态细胞的突变体病毒感染病毒感染SWA病毒
公开(公告)号：WO2010135054A2
公开(公告)日：2010-11-25
申请号：PCT/US2010/032151
申请日：2010-04-23
申请人： THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF AGRICULTURE , BORCA, Manuel, V. , ZHU, James, J.
发明人： BORCA, Manuel, V. , ZHU, James, J.
IPC分类号： C12N15/40 , C12N15/63 , C12N7/01 , C07K14/08 , A61K39/12
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Brescia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（源自含有高毒力菌株Brescia的遗传信息的感染性克隆），其含有位置2566,2567和2568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。

8. 发明申请

WO2010047955A2 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：经典猪发热病毒和新型经典猪发疹病毒E0和E2糖蛋白中N-连接的糖基化修饰
公开(公告)号：WO2010047955A2
公开(公告)日：2010-04-29
申请号：PCT/US2009/059920
申请日：2009-10-08
申请人： THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF AGRICULTURE , BORCA, Manuel, V. , RISATTI, Guillermo, R.
发明人： BORCA, Manuel, V. , RISATTI, Guillermo, R.
IPC分类号： C12N15/33 , C12N7/00 , A61K39/12
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是经典猪瘟病毒（CSFV）的三种包膜糖蛋白中的一种。 E2涉及几种功能，包括病毒附着和进入靶细胞，产生抗体，诱导猪的保护性免疫应答和毒力。 E2中7个推定的糖基化位点通过CSFV Brescia感染性克隆（BICv）的定点诱变进行修饰。获得一组病毒突变体并用于研究E2糖蛋白中推定的糖基化位点的去除是否会影响猪的病毒毒力/发病机制。我们观察到拯救存活病毒完全受到E2中所有推定的糖基化位点的去除的损害，但当突变N185A恢复为野生型天冬酰胺产生在猪中减毒的活的病毒时恢复。每个E2糖基化位点的单个突变显示氨基酸N116（N1v病毒）负责BICv的减毒。 N1 v在感染后第3天和第28天有效保护了猪免于攻击强毒BICv，这表明可以修改E2的糖基化以开发CSF活减毒疫苗。此外，一种新开发的病毒，包含E2中假定的糖基化位点N1和EO（6b）中的N 1缺失，称为N1 E0 / 2v，在接种后第3天和第28天诱导针对攻击的固体保护。

9. 发明申请

WO2009091720A2 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：经典蛇型病毒和新型经典开花病毒疫苗的E1糖蛋白中的N-连接的糖苷化改变
公开(公告)号：WO2009091720A2
公开(公告)日：2009-07-23
申请号：PCT/US2009/030824
申请日：2009-01-13
申请人： THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF 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 Erπs 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： E1，以及Ems和E2是经典猪瘟病毒（CSFV）的三种信封糖蛋白之一。我们以前的研究表明，E2或Erns的糖基化状态强烈影响猪的病毒毒力。在这里，我们调查了在自然宿主感染期间高毒性CSFV株Brescia的E1糖基化作用。通过CSFV布雷西亚感染性克隆（BICv）的定点诱变修饰E1中三个推定的糖基化位点。获得一组病毒突变体，用于检测E1糖蛋白中推定的糖基化位点的去除是否会影响猪的病毒毒力/发病机制。我们观察到，通过去除E1中所有三个推定的糖基化位点，完全损害了存活的病毒的拯救。每个E1糖基化位点的单突变显示CSFV氨基酸N594（E1.N3病毒）以及N500和N513（E1.N1N2病毒）的组合突变导致BICv衰减。感染E1。 N1N2或E1.N3病毒能够在感染后3和28天有效地保护猪免受毒性BICv的攻击。这些结果以及显示Erps和E2糖基化作用的结果表明，糖基化模式的操作可能是开发CSF减毒疫苗的有用工具。

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