Immunobiology Of Scrapie Virus Infection

绵羊瘙痒病是一组疾病的原型，统称为传染性海绵状脑病(TSE)。TSE疾病影响的物种范围很广，包括羊、牛、水貂、人、鹿、麋鹿等。该组织的最新成员牛海绵状脑病(BSE)或疯牛病被认为来自感染羊痒病的绵羊。没有证据表明羊痒病会感染人类。然而，羊瘙痒病通过牛的传播似乎改变了它的生物学特性，使疯牛病可以传播给人类。TSE疾病的种间传播很难预测，但可能涉及所研究物种的特定蛋白质分子之间的复杂相互作用，特别是一种名为普恩蛋白(PrP)的蛋白质。该项目的总体目标是确定PrP和其他因素和条件如何管理从一个物种到另一个物种的传播。将根据这些发现制定干预传播或疾病过程的战略。现已知，不同物种之间以及不同物种之间的TSE疾病的跨物种传播涉及所涉及物种的PrP之间的相互作用。例如，在少数情况下，引起老鼠疾病的TSE制剂可以引起仓鼠的疾病，但仓鼠制剂不会引起老鼠的临床疾病。为了确定PrP如何影响从仓鼠到老鼠的传播，我们开发了表达仓鼠PrP(HPRP)的转基因小鼠。HPRP的表达有多种启动子，包括神经元特异性的烯醇化酶启动子(NSE)，星形胶质细胞的GFAP特异性启动子，以及在许多组织中导致表达的天然启动子。这些类型的小鼠中的每一种都被培育成PrP缺失的小鼠，这些小鼠不表达小鼠PrP。因此，我们有几种类型的小鼠，我们已经用来研究PrP在特定细胞类型中的表达如何影响对瘙痒病的敏感性，以及HPRP和小鼠PrP(MoPrP)是如何相互作用的。在自然启动子或NSE启动子控制下表达HPRP的小鼠在脑内接种后对仓鼠瘙痒病完全敏感。这一结果表明PrP对传递至关重要，仅限于神经元的表达就足以进行传递。如果在缺乏MoPrP功能表达的小鼠中表达NSE/HPRP，潜伏期就会缩短，这表明MoPrP的存在在某种程度上与HPRP竞争，从而延迟了疾病的发生。这种效果是对小鼠的保护作用，因为这种疾病要么被推迟，要么完全被绕过。在GFAP启动子(GFAP/HPRP)控制下表达HPRP的小鼠在接种仓鼠瘙痒病制剂后没有出现临床疾病。然而，如果GFAP/HPRP在缺乏MoPrP表达的小鼠中表达，那么它们在接种仓鼠制剂后确实会生病。这一结果表明，星形胶质细胞特异性表达HPRP也足以进行传递，尽管比仅限于神经元表达的效率要低得多。此外，在表达MoPrP和HPRP的小鼠中，这一结果表明MoPrP和HPRP之间存在非常强的干扰。由于TSE疾病被认为是通过口服传播的，我们还将HPRP在多个组织中表达的另一个TG系Tg7和NSE/HPRP TG小鼠接种到仓鼠瘙痒病疫苗的口服和腹腔注射中。通过这些途径接种的小鼠，如果同时表达小鼠和HPRP，大多数都能存活下来。这些结果表明，对TSE疾病的治疗干预可能基于这些干扰机制。我们还发现，仓鼠瘙痒病制剂在正常小鼠(被认为对仓鼠瘙痒病具有抵抗力)中持续存在，持续时间长于预期寿命，即使小鼠没有瘙痒病症状。这种持久性依赖于PrP的存在。我们还测定了仓鼠剂从小鼠体内清除的动力学。我们的研究表明，尽管大多数病原体被迅速淘汰，但存留的病原体最终可以适应新的物种，尽管需要盲目进入第二个受体群体。从第二代小鼠获得的脑和脾匀浆也在仓鼠和小鼠身上引起了临床疾病(第三代)。因此，仓鼠制剂在一个被认为具有抵抗力的物种中至少坚持了三次。第三代小鼠的潜伏期因供者不同而不同。我们相信，在第三个阶段，我们开始看到“菌株”的发展。这一结果与牛疯牛病起源于绵羊的理论相一致，但在牛身上并没有表现为临床疾病，直到牛发生了一次以上的初级传代。对抗性物种的持久性和最终适应表明，类似的情况也可能发生在其他物种的组合中。在美国，人们担心鹿和麋鹿的慢性消耗性疾病可能会传播给包括人类在内的其他物种，尽管宿主范围尚未完全划定。我们正在利用各种技术来研究这个问题。

Scrapie of sheep is the prototype of a group of diseases collectively designated transmissible spongiform encephalopathies (TSE). TSE diseases affect a wide range of species including sheep, cattle, mink, humans, deer, elk and others. The newest member of the group, bovine spongiform encephalopathy (BSE) or mad cow disease is believed to have been derived from scrapie infected sheep. There is no evidence that sheep scrapie infects humans. However, passage of sheep scrapie through cattle appears to have altered its biological characteristics making BSE transmissible to humans. Interspecies transmission of TSE diseases is difficult to predict but probably involves complex interactions between specific protein molecules of the species considered and in particular a protein designated prion protein (PrP). The overall objective of this project is to determine how PrP and other factors and conditions govern transmission from one species to another. Strategies to interfere with the transmission or disease process will be developed based on these findings. Interspecies transmission of TSE diseases between and among various species is now known to involve interactions between the prion proteins (PrP) of the species involved. For example, the TSE agent which causes disease in mice can in a few circumstances cause disease in hamsters but the hamster agent does not cause clinical disease in mice. To determine how PrP influences transmission from hamsters to mice we have developed transgenic mice which express hamster prion protein (HPrP). The HPrP has been expressed using a variety of promoters including the neuron- specific enolase promoter (NSE) which targets expression to neurons, the GFAP specific promoter to target expression to astrocytes and natural promoters which result in expression in many tissues. Each of these types of mice has been bred to PrP null mice which do not express mouse PrP. Thus, we have available several types of mice which we have used to investigate how expression of PrP in specific cell types influences susceptibility to scrapie and how HPrP and mouse PrP (MoPrP) interact. Mice which express HPrP under the control of natural or NSE promoters are completely susceptible to hamster scrapie agent following intracerebral inoculation. This result showed that PrP is critical to transmission and that expression restricted to neurons is sufficient for transmission to occur. If NSE/HPrP was expressed in mice which lacked functional MoPrP expression, the incubation period was reduced indicating that the presence of MoPrP in some way competed with HPrP to delay the onset of disease. The effect was protective for the mice in that disease was either delayed or completely circumvented. Mice which express HPrP under the control of the GFAP promoter (GFAP/HPrP) did not become clinically sick after inoculation of hamster scrapie agent. However, if GFAP/HPrP was expressed in mice which lacked MoPrP, expression they did become sick following hamster agent inoculation. This result suggested that astrocyte specific expression of HPrP was also sufficient for transmission to occur though much less efficiently than was true if expression was limited to neurons. Furthermore, this result demonstrated very strong interference between MoPrP and HPrP in the mice which expressed both. Because TSE diseases are thought to be transmitted orally we also inoculated the NSE/HPrP Tg mice and another Tg line designated Tg7, where HPrP is expressed in multiple tissues, with hamster scrapie agent orally and intraperitoneally. Most of the mice inoculated by these routes survived if they expressed both mouse and HPrP. These results suggested that therapeutic intervention in TSE diseases could be based on these interference mechanisms. We also found that hamster scrapie agent persisted in normal mice, (thought to be resistant to hamster scrapie), over their expected lifespans even though the mice remained free of scrapie symptoms. This persistence was dependent on the presence of PrP. We also determined the kinetics of hamster agent clearance from mice. We showed that although most agent is rapidly eliminated that which persists can eventually adapt to the new species though blind passage to a second recipient group was required. Brain and spleen homogenates obtained from the second pass mice also caused clinical disease in hamsters as well as mice ( 3rd pass). Thus hamster agent has persisted through at least three passes in a species thought to be resistant. Incubation periods in the third pass mice varied depending on specific donors. We belive that at the third pass we are beginning to see development of "strains". This result is consistent with the theory that BSE was derived from sheep but was not manifest as clinical disease in cattle until more than a primary pass in cattle had occurred. Persistence and eventual adaptation to a resistant species suggests that similar situations could occur in other species combinations. In the USA there is concern that chronic wasting disease of deer and elk could be transmissible to other species including humans though the host range has not yet been fully delineated. We are utilizing a variety of techiques to study this question.