Immunobiology Of Scrapie Virus Infection

羊痒病是一组统称为传染性海绵状脑病（TSE）的疾病的原型。TSE疾病影响广泛的物种，包括羊、牛、水貂、人类、鹿、麋鹿和其他物种。这一群体的最新成员，牛海绵状脑病（BSE）或疯牛病被认为是来自感染痒病的羊。没有证据表明羊痒病会感染人类。然而，羊痒病通过牛的传播似乎改变了其生物学特性，使疯牛病传播给人类。TSE疾病的种间传播很难预测，但可能涉及所考虑物种的特定蛋白质分子之间的复杂相互作用，特别是一种称为朊病毒蛋白（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控制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策略。目前已知，TSE疾病在不同物种之间和物种间的种间传播涉及相关物种的朊病毒蛋白（PrP）之间的相互作用。例如，在小鼠中引起疾病的TSE制剂在少数情况下可以引起仓鼠的疾病，但仓鼠制剂不会引起小鼠的临床疾病。为了确定鼠朊病毒如何影响仓鼠向小鼠的传播，我们开发了表达仓鼠朊病毒蛋白（HPrP）的转基因小鼠。HPrP的表达使用多种启动子，包括神经元特异性烯醇酶启动子（NSE），其目标是神经元表达，GFAP特异性启动子的目标是星形胶质细胞表达和自然启动子导致许多组织的表达。这些类型的小鼠都被培育成不表达小鼠PrP的PrP缺失小鼠。因此，我们有几种类型的小鼠，我们用它们来研究PrP在特定细胞类型中的表达如何影响痒病的易感性，以及HPrP和小鼠PrP （MoPrP）如何相互作用。在天然启动子或NSE启动子控制下表达HPrP的小鼠在脑内接种后对仓鼠痒病剂完全易感。这一结果表明，PrP对传递至关重要，并且仅限于神经元的表达足以使传递发生。如果在缺乏功能性MoPrP表达的小鼠中表达NSE/HPrP，则潜伏期缩短，这表明MoPrP的存在以某种方式与HPrP竞争以延迟疾病的发生。这种效果对小鼠是有保护作用的，因为这种疾病要么被推迟，要么被完全避免。在GFAP启动子（GFAP/HPrP）控制下表达HPrP的小鼠接种仓鼠痒病剂后未出现临床发病。然而，如果GFAP/HPrP在缺乏MoPrP的小鼠中表达，则小鼠在接种仓鼠剂后确实会发病。这一结果表明，星形胶质细胞特异性表达HPrP也足以发生传播，尽管其效率远低于仅限于神经元的表达。此外，这一结果表明，在表达mprp和HPrP的小鼠中，mprp和HPrP之间存在非常强的干扰。由于TSE疾病被认为是通过口腔传播的，我们还对NSE/HPrP Tg小鼠和另一个Tg系Tg7进行了接种，其中HPrP在多个组织中表达，并口服和腹腔注射了仓鼠痒病剂。通过这些途径接种的小鼠，如果同时表达小鼠和HPrP，大多数小鼠存活。这些结果提示可以基于这些干扰机制对TSE疾病进行治疗干预。我们还发现，仓鼠痒病制剂在正常小鼠（被认为对仓鼠痒病有抵抗力）的预期寿命内持续存在，即使小鼠仍然没有痒病症状。这种持续性依赖于PrP的存在。我们还测定了小鼠清除仓鼠剂的动力学。我们发现，虽然大多数病原体被迅速消灭，但仍然存在的病原体最终可以适应新物种，尽管需要盲传到第二个受体群体。从第二代小鼠获得的脑和脾脏匀浆也在仓鼠和小鼠（第三代）中引起临床疾病。因此，仓鼠剂在一个被认为具有抗药性的物种中至少持续存在了三次。第三代小鼠的潜伏期取决于特定的供体。我们认为，在第三阶段，我们开始看到“紧张”的发展。这一结果与疯牛病源自绵羊的理论是一致的，但在牛中出现不止一次的初级传播之前，疯牛病并未表现为牛的临床疾病。对抗性物种的持续和最终适应表明，类似的情况可能发生在其他物种组合中。在美国，人们担心鹿和麋鹿的慢性消耗性疾病可能会传染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.