CN1165342C - 猪生殖和呼吸综合症疫苗 - Google Patents
猪生殖和呼吸综合症疫苗 Download PDFInfo
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- CN1165342C CN1165342C CNB971941092A CN97194109A CN1165342C CN 1165342 C CN1165342 C CN 1165342C CN B971941092 A CNB971941092 A CN B971941092A CN 97194109 A CN97194109 A CN 97194109A CN 1165342 C CN1165342 C CN 1165342C
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Classifications
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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Abstract
本发明公开了治疗猪生殖和呼吸综合症的疫苗和方法。该疫苗衍生于病毒因子NEB-1-P94，此病毒以登记号VR-2525保藏于美国典型培养物保藏中心。此外，本发明公开了一种表型特征可以区别于野生型PRSS病毒的疫苗病毒。
Description
发明背景
本发明涉及治疗猪生殖和呼吸综合症(PRRS)的疫苗。
1987年，美国的养猪业经受了对养猪业产生严重经济影响的一种不为人知的传染性疾病。该疾病综合症在欧洲包括德国、比利时、荷兰、西班牙和英国均有报道。
该疾病特征在于生殖力衰退、呼吸疾病以及包括食欲不振、发热、呼吸困难及轻微神经病学症状的各种临床症状。所述综合症的一个主要部分为生殖力衰退，表现为早产、后期流产、初生小猪体弱、死胎、干瘪胎儿、下崽率下降和延迟发情期的恢复。呼吸疾病的临床症状在3周龄以下的小猪中最明显，但据报告存在于各个养殖阶段的猪中。受感染的小猪生长缓慢，皮毛粗糙，呼吸窘迫(“发出低沉的声音(thumping)”)及死亡率增加。
该病综合症曾以许多不同的术语提到，包括神秘的猪病(MSD)，猪流行性流产和呼吸综合症(PEARS)，猪不育和呼吸综合症(SIRS)。目前通常使用的名称为猪生殖和呼吸综合症(PRRS)；在本专利申请的全过程中将使用该术语。
本发明的目的之一是提供疫苗，该疫苗保护猪不受由PRRS引起的临床疾病影响。另一个目的是提供疫苗，该疫苗给予饲养的猪群时，将减少其群体中PRRS的存在。
发明概述
本发明的目的之一是提供一新疫苗，该疫苗保护猪免受由猪生殖和呼吸综合症(PRRS)病毒引起的临床疾病。
本发明再一目的是提供疫苗，该疫苗保护猪不受PRRS病毒NEB-1株的影响。
本发明再一目的是提供保护猪免受由猪生殖和呼吸疾病病毒引起的临床疾病的方法。
本发明的再一目的是提供疫苗病毒，它具有可以与野生型PRRS病毒相区别的表型特征。
发明详述
本发明提供包含一种减毒的猪呼吸和生殖综合症(PRRS)病毒的物质组合物，该病毒经实验室处理而修饰，以用于疫苗接种。此外，该组合物具有允许它用于诊断目的的表型特征，从而可对已经自然感染了PRRS病毒的猪和仅仅接触疫苗毒株的动物进行区别。PRRS病毒分离株NEB-1-P94已于美国典型培养物保藏中心保藏(登记号为VR-2525)。
PRRS病毒的毒性分离株由来自Nebraska大学诊断实验室的一头死猪的组织样本中获得。将来自该死猪的组织匀浆液接种在原代猪肺泡巨噬细胞上，并通过对接种培养物而非对照培养物的细胞致病作用来检测病毒的存在。随后基于物理特性(对醚和氯仿的敏感性、浮力密度和缺乏血细胞凝集活性)、与特定抗体的反应性及遗传分析，将该分离病毒(称为NEB-1)特征鉴定为PRRS病毒。将该病毒接种于饲养的小猪中产生以高热、呼吸改变和与病毒间质性肺炎一致的肺部病状为特征的呼吸疾病。另外，将该病毒接种于怀孕母猪中产生生殖疾病，其特征为胎儿干瘪、产死小猪和小猪初生体弱以致随后死亡。由这种病毒引起的呼吸和生殖疾病是所报告的PRRS病毒综合症的特征。
将NEB-1病毒在组织培养中经连续传代减毒。首先将该病毒接种于原代猪肺泡巨噬细胞(SAM)培养物中而使病毒传代(头两代)，然后在MA104细胞(可从Microbiological Associates，Inc.，Rockville，MD购得)上进行连续传代至总共94代。在此过程中，经空斑纯化及鉴定表型特性的特征而分离病毒克隆。被称为NEB-1-P94的疫苗克隆因在猪肺泡巨噬细胞上生长减弱而被选择出来，它缺乏与PRRS特异性单克隆抗体SDOW 17(ATCC HB10997)的反应性，缺乏对小猪和怀孕母猪的疾病的诱导作用。将NEB-1-P94在MA104细胞上扩增并作为主种子病毒(也称为PRRS-MSV-94-1)进行冷冻以用于疫苗开发研究。
应用MA104细胞作为基质制备疫苗(不过也可应用另外的、可支持PRRS病毒生长的细胞系等，例如MARC145细胞[可从Dr.Wang，Agriculture Research Station，Clay Center NE处获得]。MA104细胞在例如850cm2的园筒形瓶的合适组织培养容器中，用含5％-10％牛血清、30mM HEPES(N-2-羟乙基哌嗪-N’-2-乙磺酸)、2mM L-谷酰胺和抗生素(例如30μg/ml庆大霉素)的Eagle氏极限必需培养基(EMEM)使其生长至连合。也可应用可以支持MA104细胞生长的其它组织培养基，例如Dulbecco氏改进必需培养基(DMEM)、培养基199或其他培养基。用NEB-1-P94病毒以1∶5-1∶1000、最好为1∶10的感染复数接种连合的单层MA104细胞。接种后在37℃培养3-5天，通过倾析收集培养上清液。
在上述加富的EMEM中制备系列稀释液，并以每孔0.2ml接种96孔组织培养板的至少4个重复孔内的连合MA104或MARC145细胞，以对病毒液进行效价测定。培养基在37℃，3-5％CO2的潮湿室内培养5天并观察其细胞病变效应。按照Spearman和Karber的方法(Methods in Virology，第IV卷，K.Maramorosch和H.Koprowski(编辑)，Academic Press，纽约，1977)计算效价(50％终点)。可以用80％丙酮固定细胞，并测试与SDOW17的反应性的缺乏和与V017或EP147(可从Dr.E.Nelson，South Dakota State University，Brookings，SD处获得)单克隆抗体呈阳性反应性(预期为阳性结果)，以确证该病毒的表型特性。
为了制备灭活疫苗，将病毒液与化学灭活剂共培育。灭活剂的实例包括甲醛、戊二醛、二元氮丙啶或β-丙醇酸内酯。然后将病毒液在4℃贮藏直至配制为疫苗。将病毒液(基于灭活前效价，含106-109 TCID50的病毒)与生理上可接受的稀释液(例如EMEM、Hank氏平衡盐溶液、磷酸盐缓冲盐水)以及免疫刺激佐剂(例如矿物油、植物油、氢氧化铝、皂角苷、非离子去垢剂、角鲨烯、嵌段共聚物或其它本领域熟知的化合物，单独或混合使用)混合以制备疫苗。疫苗剂量通常为1-5ml。
关于活疫苗制剂，将病毒液在-50℃或以下冷藏直至使用。将范围为104.0-107.0TCID50/剂量、最好为含106.0TCID50/剂量的病毒液用生理上可适用的稀释液(例如EMEM、Hank氏平衡盐溶液、磷酸盐缓冲盐水)和生理上可适用的设计用来稳定该病毒的混合物稀释。本领域已知的可以单独应用或联合应用以稳定该病毒的组合物稀释。本领域已知的能够单独应用或联合应用以稳定病毒的化合物包括蔗糖、乳糖、N-Z胺、谷胱甘肽、新蛋白胨、明胶、葡聚糖和胰蛋白胨、将疫苗冷冻贮藏(-50℃或更冷)或冻干于4℃贮藏直至使用。该疫苗的剂量大小范围通常为1-5ml，最好为2ml。
为预防PRRS诱导的疾病，可经口服、鼻内给药或非肠道给药的方式给与猪该疫苗。非肠道给药途径的实例包括皮内、肌内、静脉内、腹膜内和皮下给药途径。
当以溶液形式给药时，可将本疫苗制成水溶液、糖浆、酏剂或酊剂形式。这样制剂在本领域是已知的，并通过将抗原和其它合适的添加剂溶解在合适的溶剂系统中而制备。这些溶剂包括水、盐水、乙醇、乙二醇、甘油、Al流体等。本领域已知的合适添加剂包括合格的染料、香料、甜味剂和抗微生物防腐剂，如硫柳汞(乙基汞硫代水扬酸钠)。这些溶液可例如经加入部分水解的明胶、山梨醇或细胞培养基而得到稳定，并可用本领域已知的方法，用本领域已知的试剂，如磷酸氢二钠、磷酸二氢钠、磷酸氢二钾和/或磷酸二氢钾使这些溶液得到缓冲。
液体制剂也可包括悬液和乳液。如使用胶体磨制备悬液，及如使用匀浆器制备乳液在本领域是已知的。
设计用来注入体液系统的非肠道剂量形式要求有合适的等渗性和缓冲至与猪体液相应水平的pH。非肠道制剂在使用前也必须先灭菌。
可用氯化钠和其它所需要的盐对等渗性进行调节。可以用其它的溶剂(如乙醇或丙二醇)来增加该组合物成分的溶解度及该溶液的稳定性。此外在本制剂中可以应用的添加剂包括葡萄糖、常规抗氧化剂和常规鳌合剂，如乙二胺四乙酸(EDTA)。
初次免疫之后2-4周可以给与加强疫苗接种。为了预防生殖性疾病，通常进行该疫苗接种法直至配种前6周和配种后1周。为了预防小猪的呼吸疾病，早在小猪3周龄时即给予疫苗接种。应用酶联免疫吸附测定(ELISA)、血清中和分析、间接免疫荧光或Western印迹法测定针PRRS病毒的抗体效价，可以监测免疫接种的反应。
该疫苗株具有的表型特性，可以用来诊断受野生型PRRS感染的猪和仅仅暴露于该疫苗株的猪。通过测定抗体与单克隆抗体(Mab)SDOW17识别的抗原决定基的反应，可以将接触PRRS病毒现场毒株的牲畜与仅接触NEW-1-P94疫苗株的牲畜区别开来。与SDOW17抗原决定基起反应抗体的存在表示出曾暴露于野生型病毒。
针对SDOW1 7抗原决定基的抗体的测定可用竞争ELISA法完成。将板(96孔)用NEB-1 PRRS病毒(或表达SDOW17抗原决定基的其它PRRS病毒)包被。然后将板与来自实验动物的猪血清和酶标记(例如与辣根过氧化酶结合的)的SDOW17单克隆抗体温育。通过缺乏酶底物颜色变换检测的酶联SDOW17 Mab与该板结合的抑制，检测猪血清识别SDOW17抗原决定基的能力。或者，可以采用直接ELISA法。包含SDOW17抗原决定基的氨基酸序列可作为合成肽而制备，或在如大肠杆菌的合适载体系统中用重组DNA表达的方法制备。将包被SDOW17抗原的板与猪血清进行温育。通过其与酶联抗猪免疫球蛋白抗血清的温育、随后与酶底物进行温育并检测颜色变化，可对猪抗体与SDOW17抗原的结合进行检测。
以下实施例详细描述本发明。在不偏离本发明目的和意义的情况下，可以对材料和方法实施修改，这对于本领域技术熟练人员来说是显而易见的。
实施例1
NEB-1-P94在肺泡巨噬细胞上生长的表型表征
将从标准种起第五代的NEB-1-P94疫苗株病毒对在MA104、MARC145和猪肺泡巨噬细胞上的生长进行特征描述。用盐水进行支气管肺泡灌洗、接着离心使细胞沉淀以获得猪肺泡巨噬细胞(SAM)。将巨噬细胞重悬浮于具有10％胎牛血清和50μg/ml庆大霉素的EMEM中，将其以每孔大约7×104个细胞的量铺平板于96孔组织培养板。将MA104细胞和MARC 145细胞铺平板于96孔组织培养板内的培养基(含10％胎牛血清、30mM HEPES、2mM L-谷酰胺和50μg/ml庆大霉素的EMEM)中。将NEB-1-P94或该NEB-1亲代病毒在培养基中进行系列稀释，将0.2ml的每种稀释液接种于含SAM、MA104或MARC145细胞的96孔板的重复孔内。培养物在37℃、3％-5％CO2的潮湿室内培养5天并监测典型PRRS病毒的细胞病变效应。根据Spearman和Karber的方法计算效价(50％终点)。与在MA104和MARC 145细胞上获得的效价相比，NEB-1-P94在三个独立的SAM培养物中显示出效价降低或无法测到效价(表1)。与在所有测试的培养物上显示出相似效价的亲代株相比，这是一种表型改变。所以，在猪肺泡巨噬细胞上生长减弱是疫苗株NEB-1-P94的一种选择性表型标记。
表1.NEB-1-P94病毒在各种细胞类型上生长的比较
| 病毒 | 在MA104上的效价* | 在MARC 145上的效价* | 在SAM 7上的效价* | 在SAM 8上的效价* | 在SAM 22上的效价* |
| NEB-1-P94 | 5.3 | 6.1 | ≤1.2 | 2.5 | ＜1.2 |
| NEB-1 | 5.2 | 6.5 | 5.2 | 6.5 | 6.5 |
*效价＝log10 TCID50/ml(在本测定中的检测极限＝1.2)
实施例2
NEB-1-P94缺乏对猪的毒性的表型特征描述
将PRRS血清反应阴性的母猪生育的四头限菌小猪(年龄为7-10天)鼻内接种(3ml/鼻孔)NEB-1-P94标准种子病毒(105.3TCID50/ml)。观察小猪呼吸疾病的临床症状，并将该疫苗病毒从接种后5天时的血清再分离出来。用第一组猪的血清鼻内接种限菌的第二组猪，并以同样的方式监测第二组猪。在小猪中重复此过程，总共连续回接传代五次，以确定是否该疫苗株能够回复为毒性状态。从每次连续的动物传代中回收疫苗病毒，然而，在限菌猪中没有观察到呼吸疾病。另外，将从第五次回接传代的猪中分离出的病毒鼻内接种入一周大和三周大的常规猪中(每只小猪给予大约105.3TCID50/ml)。病毒接种后观察动物42天，没有发现与毒性PRRS感染一致的临床疾病症状(如延续高热、呼吸症状、肺损伤)。所以，对NEB-1-P94病毒作出结论：在小猪中诱导呼吸疾病方面，它是无毒性的。
接下来是检测NEB-1-P94病毒引起生殖疾病的能力。用标准种子疫苗株(104.5TCID50/ml)鼻内接种(3ml/鼻孔)妊娠85天的PRRS血清反应阴性的母猪。所有母猪在预期时间生崽，并且96％的小猪出生时存活并健康。相比较，未接种的对照母猪一胎产生的小猪有87％存活并且健康。所以，疫苗株NEB-1-P94不能诱导毒性PRRS病毒所引起的典型生殖疾病(见实施例4)。这些数据确证了疫苗株NEB-1-P94的无毒性表型。
实施例3
NEB-1-P94与PRRS病毒特异性单克隆抗体
反应性的表型特性描述
以间接免疫荧光法测定感染亲株NEB-1病毒或疫苗株NEB-1-P94病毒的MA104细胞与PRRS病毒特异性单克隆抗体的反应性。简短地说，感染各种病毒2天后，用80％丙酮对96孔板的连合MA104细胞固定10分钟。然后将单层细胞与SDOW17、V017或EP147单克隆抗体温育。洗涤后，与结合异硫氢酸荧光素的抗小鼠IgG温育、洗涤并通过显微镜测定荧光，以检测单克隆抗体与每种病毒的反应性。周所有3种单克隆抗体对于NEB-1亲株都表现出荧光阳性(表2)。然而，疫苗株NEB-1-P94已经丧失了与SDOW17单克隆抗体的反应性，但是与其它两种单克隆抗体则呈阳性。这些数据表明：该NEB-1-P94株已经丧失了SDOW17抗体识别的抗原决定基的表达。与该单克隆抗体反应性的丧失很可能体现了NEB-1-P94的RNA序列中的遗传突变，该突变导致SDOW17识别的核衣壳蛋白区域的氨基酸序列发生改变。
表2.PRRS亲株和PRRS疫苗株与特异性单克隆抗体的反应性
| 与SDOW1 7的反应性 | 与VO17的反应性 | 与EP147的反应性 | |
| NEB-1-P94 | - | + | + |
| NEB-1 | + | + | + |
实施例4
用NEB-1-P94接种母猪以预防生殖性疾病
在含10％胎牛血清、2mM L-谷酰胺和30μg/ml庆大霉素的EMEM中，用NEB-1-P94(从标准种子经4次传代)以大约1∶10的感染复数接种圆筒形瓶中的连合MA104细胞以制备疫苗。培养物在37℃培养3天，然后通过倾析收集上清液。病毒液用稳定剂(75g/L胰蛋白胨、30g/L葡聚糖、2g/L明胶、100g/L乳糖、2g/L谷氨酸钠、1.05g/LKH2PO4、2.5g/L K2HPO4、10g/L白蛋白组分V)稀释50％(V/V)，冷冻并冻干。疫苗用无菌去离子水再水化，在配种前4-6周取2ml(105.1TCID50/ml)肌内给予小母猪。
妊娠85天时，鼻内给予NEB-1病毒(大约106.3 TCID50)，对已接种疫苗的小母猪和未接种疫苗的对照小母猪进行攻击。监测动物生崽后整个七周内因PRRS病毒造成的胎儿或新生小猪死亡的情况。在11/12(92％)的对照母猪和100％的这些母猪生的、出生时存活的小猪中出现了PRRS病毒血症。在妊娠期间感染PRRS导致在对照组中16％的分娩死亡(大型干尸和死胎猪)(表3)，相比较，在接种了疫苗的母猪中只有6％死亡。另外，和对照组相比，接种疫苗可使体弱和站立不稳小猪的发生率降低50％，并导致出生时体重低(出生时体重小于2磅)的小猪减少94％。和对照组相比，在免疫母猪产的所有小猪的血液或组织中没出现PRRS病毒，并且在出生7周内防止了55％的死亡损失(表3)，证明接种疫苗预防了先天的PRRS。在接种了疫苗的母猪和它们的后代中出现的对死亡损失和病毒感染的统计显著性预防作用，清楚地证明该疫苗对预防生殖形式的PRRS病毒诱导的疾病方面的效力。
表3.PRRS病毒攻击接种疫苗母猪与对照母猪后，表现出的生殖性疾病的概述
| 组别 | 母猪数(猪平均数/胎) | 大型干尸和死产％ | 出生存活但随后死亡％ | 体弱及站立不稳％ | 小猪体重＜2Ibs.％ | 7周死亡率％ |
| 接种疫苗组 | 21(10.8) | 6％ | 2％ | 3％ | 1％ | 17％ |
| 对照组 | 12(10.1) | 16％ | 3％ | 6％ | 17％ | 38％ |
实施例5
用NEB-1-P94接种母猪以预防呼吸性疾病
在含10％胎牛血清、2mM L-谷酰胺和30μg/ml庆大霉素的EMEM中，用NEB-1-P94(从标准种子传至第4代)以大约1∶10的感染复数接种圆筒形瓶中的连合MA104细胞以制备疫苗。在37℃将培养物培养5天，然后通过倾析收集上清液。病毒液用稳定剂(75g/L胰蛋白胨、30g/L葡聚糖、2g/L明胶、100g/L乳糖、2g/L谷氨酸钠、1.05g/L KH2PO4、2.5g/L K2HPO4、10g/L白蛋白组分V)稀释50％(v/v)，冷冻并冻干。疫苗用无菌去离子水再水化，取2ml(105.1TCID50/ml)肌内给予PRRS血清反应阴性的3周龄小猪。
接种后4周，用毒性NEB-1-P94病毒经所述用于小母猪(实施例4)的鼻内给药途径攻击小猪。攻击后监测14天小猪呼吸性疾病的症状。所有未接种疫苗的对照小猪都出现了呼吸性疾病的临床症状，相比较，接种了疫苗的动物只有3/40(8％)。和对照相比，在接种了疫苗的动物中，疫苗接种导致发热、呼吸症状和临床疾病在统计学上明显地减少(表4)。本研究清楚地证明在小猪中该疫苗对预防PRRS病毒诱导的呼吸疾病的效力。
表4.在PRRS病毒攻击后的疫苗接种动物和对照动物中临床疾病的总结
| 在所示天数时具有临床症状的动物的百分比 | |||||||||||||||
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
| 呼吸症状 | |||||||||||||||
| 对照 | 0 | 0 | 0 | 57 | 53 | 37 | 60 | 40 | 37 | 40 | 47 | 40 | 23 | 30 | 33 |
| 接种疫苗 | 0 | 0 | 0 | 8 | 8 | 0 | 2 | 8 | 0 | 2 | 5 | 5 | 5 | 2 | 0 |
| 发热＞104.5°F | |||||||||||||||
| 对照 | 3 | 17 | 43 | 73 | 83 | 73 | 66 | 50 | 37 | 57 | 47 | 37 | 20 | 17 | NA |
| 接种疫苗 | 0 | 14 | 20 | 20 | 14 | 10 | 10 | 20 | 2 | 14 | 10 | 10 | 4 | 4 | NA |
| 临床疾病 | |||||||||||||||
| 对照 | 0 | 0 | 0 | 23 | 60 | 50 | 60 | 47 | 33 | 50 | 50 | 40 | 33 | 27 | 33 |
| 接种疫苗 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 2 | 0 | 2 | 2 | 2 | 0 | 0 | 0 |
NA＝数据不可利用
对照n＝30
疫苗接种n＝40
Claims (7)
1.一种适合用于保护猪以抵抗猪的生殖和呼吸综合症的疫苗，该疫苗包含NEB-1-P94分离株，保藏于美国典型培养物保藏中心，登记号为VR-2525。
2.权利要求1的疫苗、其中该分离株是以活的、灭活的或减毒的形式。
3.权利要求2的疫苗，其中该分离株是以减毒的形式并为液体的、冷冻的或干燥的形式。
4.一种适合用于使猪免疫而抵抗猪的生殖和呼吸综合症的疫苗，该疫苗包含具有全部等同于分离株NEB-1-P94的特性的PRRS病毒，保藏于美国典型培养物保藏中心，登记号为VR-2525。
5.权利要求1或4的疫苗，该疫苗的每ml含有104.0至109.0TCID50量的该PRRS病毒。
6.权利要求1或4的疫苗的应用，该应用为将该疫苗用于制备用于保护猪使猪免受由猪的生殖和呼吸综合症病毒引起的疾病的药物。
7.权利要求6的应用，其中所述的药物是经口服、鼻内、肌内、皮内、静脉内或皮下给予的药物。
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| CN101612395B (zh) * | 2008-06-24 | 2012-02-08 | 扬州优邦生物制药有限公司 | 一种培养敏感细胞生产蓝耳病疫苗的方法 |
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| EP0839912A1 (en) | 1996-10-30 | 1998-05-06 | Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) | Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof |
| CA2290220C (en) | 1998-12-22 | 2013-11-19 | Pfizer Products Inc. | An infectious cdna clone of north american porcine reproductive and respiratory syndrome (prrs) virus and uses thereof |
| AU3198200A (en) | 1999-03-08 | 2000-09-28 | Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. | Prrsv vaccines |
| DK2251419T3 (da) | 1999-04-22 | 2012-07-02 | Us Agriculture | Porcint reproduktions- og respirations-syndrom-vaccine baseret på isolat JA-142 |
| CA2571452A1 (en) | 2004-06-18 | 2006-01-26 | Regents Of The University Of Minnesota | Identifying virally infected and vaccinated organisms |
| US7632636B2 (en) | 2004-09-21 | 2009-12-15 | Boehringer Ingelheim Vetmedica, Inc. | Porcine reproductive and respiratory syndrome isolates and methods of use |
| PT1904631E (pt) | 2005-06-24 | 2012-07-16 | Univ Minnesota | Vírus srrp, clones infecciosos, os seus mutantes e processos de utilização |
| TWI627281B (zh) | 2009-09-02 | 2018-06-21 | 百靈佳殷格翰家畜藥品公司 | 降低ｐｃｖ－２組合物殺病毒活性之方法及具有改良免疫原性之ｐｃｖ－２組合物 |
| CN103370077B (zh) | 2011-02-17 | 2016-10-26 | 贝林格尔.英格海姆维特梅迪卡有限公司 | 猪繁殖与呼吸综合征病毒的商业化规模生产方法 |
| MY186535A (en) | 2011-02-17 | 2021-07-26 | Boehringer Ingelheim Vetmedica Gmbh | Novel european prrsv strain |
| EP2737059A1 (en) | 2011-07-29 | 2014-06-04 | Boehringer Ingelheim Vetmedica GmbH | Novel prrs virus inducing type i interferon in susceptible cells |
| US9187731B2 (en) | 2011-07-29 | 2015-11-17 | Boehringer Ingelheim Vetmedica Gmbh | PRRS virus inducing type I interferon in susceptible cells |
| KR101300528B1 (ko) * | 2011-10-05 | 2013-09-02 | 캔 테크놀로지스 인코포레이티드 | 액티노바실러스 플루로뉴모니애 감염 방지 및 처치용 조성물 |
| WO2014150822A2 (en) | 2013-03-15 | 2014-09-25 | Boehringer Ingelheim Vetmedica, Inc. | Porcine reproductive and respiratory syndrome virus, compositions, vaccine and methods of use |
| EP4012025A1 (en) * | 2020-12-10 | 2022-06-15 | Univerzita Palackého v Olomouci | Transport medium for samples containing nucleic acids and/or proteins |
| CZ309704B6 (cs) * | 2020-12-10 | 2023-08-09 | Univerzita Palackého v Olomouci | Transportní médium pro transport a skladování virů |
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| US6042830A (en) * | 1992-08-05 | 2000-03-28 | Boehringer Ingelheim Vetmedica, Inc. | Viral agent associated with mystery swine disease |
| DE69206631T2 (de) * | 1991-10-14 | 1996-05-15 | Akzo Nobel Nv | Impfstoff gegen das Fortpflanzungs- und Atmungssyndrom bei Schweinen (PRRS) und Diagnose. |
| FR2686097B1 (fr) * | 1992-01-14 | 1994-12-30 | Rhone Merieux | Preparation d'antigenes et de vaccins de virus de la mystery disease, antigenes et vaccins obtenus pour la prevention de cette maladie. |
| US5695766A (en) * | 1992-10-30 | 1997-12-09 | Iowa State University Research Foundation | Highly virulent porcine reproductive and respiratory syndrome viruses which produce lesions in pigs and vaccines that protect pigs against said syndrome |
| ES2152304T3 (es) * | 1993-02-08 | 2001-02-01 | Bayer Ag | Procedimiento para el crecimiento del virus del sindrome reproductivo y respiratorio porcino y su uso en vacunas. |
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| ATE206455T1 (de) * | 1994-04-11 | 2001-10-15 | Akzo Nobel Nv | Europäische vakzinstämme des fortplanzungs- atmungs-syndromsvirus des schweins |
| US5690940A (en) * | 1995-06-21 | 1997-11-25 | Regents Of The University Of Minnesota | Low pathogencity PRRS live virus vaccines and methods of preparation thereof |
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| CN101612395B (zh) * | 2008-06-24 | 2012-02-08 | 扬州优邦生物制药有限公司 | 一种培养敏感细胞生产蓝耳病疫苗的方法 |
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| HUP9901958A2 (hu) | 1999-10-28 |
| EE9800267A (et) | 1999-02-15 |
| BG102809A (en) | 1999-05-31 |
| CN1216922A (zh) | 1999-05-19 |
| PL328627A1 (en) | 1999-02-15 |
| MY115070A (en) | 2003-03-31 |
| RU2187333C2 (ru) | 2002-08-20 |
| EE04741B1 (et) | 2006-12-15 |
| SK119498A3 (en) | 1999-07-12 |
| HUP9901958A3 (en) | 2000-04-28 |
| BR9708443B1 (pt) | 2012-06-12 |
| BR9708443A (pt) | 1999-08-03 |
| WO1997031651A1 (en) | 1997-09-04 |
| BG64693B1 (bg) | 2005-12-30 |
| KR100297537B1 (ko) | 2001-10-26 |
| MX9807083A (es) | 1998-12-31 |
| CZ273798A3 (cs) | 1999-02-17 |
| EP0894007A1 (en) | 1999-02-03 |
| AU2277497A (en) | 1997-09-16 |
| AR006023A1 (es) | 1999-07-21 |
| ZA971663B (en) | 1997-08-26 |
| CO4600644A1 (es) | 1998-05-08 |
| CA2248182A1 (en) | 1997-09-04 |
| CA2248182C (en) | 2004-04-20 |
| KR19990087432A (ko) | 1999-12-27 |
| JP3135069B2 (ja) | 2001-02-13 |
| JPH11506122A (ja) | 1999-06-02 |
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Address after: new jersey Patentee after: Schering Corporation Address before: new jersey Patentee before: SCHERING CORP (US) |
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Granted publication date: 20040908 |