Immunobiology Of Scrapie Virus Infection

羊瘙痒病是一组统称为传染性海绵状脑病（TSE）的疾病的原型。TSE疾病影响范围广泛的物种，包括羊、牛、水貂、人类、鹿、麋鹿等。该组的最新成员，牛海绵状脑病（BSE）或疯牛病被认为是由于牛被喂食骨粉和蛋白质补充剂污染的组织从羊瘙痒症感染的羊。没有证据表明羊痒病会感染人类。然而，绵羊瘙痒病通过牛的传播似乎改变了其生物学特性，使BSE可传播给人类。很难预测哪种TSE疾病会传染给哪种其他物种，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战略。目前已知不同物种之间的TSE疾病的种间传播涉及所涉及物种的朊病毒蛋白（PrP）之间的相互作用。例如，在小鼠中引起疾病的TSE因子在少数情况下可在仓鼠中引起疾病，但仓鼠因子不会在小鼠中引起临床疾病。为了确定PrP如何影响从仓鼠到小鼠的传播，我们开发了表达仓鼠朊蛋白（HPrP）的转基因小鼠。HPrP已经使用多种启动子表达，包括靶向表达至神经元的神经元特异性烯醇化酶启动子（NSE）、靶向表达至星形胶质细胞的GFAP特异性启动子和导致在许多组织中表达的天然启动子。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，我们已经用来研究如何表达的PrP在特定的细胞类型影响羊瘙痒症的易感性，以及如何HPrP和小鼠PrP（MoPrP）相互作用。在天然或NSE启动子控制下表达HPrP的小鼠在脑内接种后对仓鼠瘙痒症因子完全敏感。这一结果表明，PrP是至关重要的传输和表达仅限于神经元是足够的传输发生。如果NSE/HPrP在缺乏功能性MoPrP表达的小鼠中表达，则潜伏期缩短，表明MoPrP的存在以某种方式与HPrP竞争以延迟疾病的发作。这种效果对小鼠是保护性的，因为这种疾病要么被推迟，要么被完全避免。在GFAP启动子控制下表达HPrP的小鼠（GFAP/HPrP）在接种仓鼠瘙痒症因子后没有临床上生病。然而，如果GFAP/HPrP在缺乏MoPrP的小鼠中表达，则它们在仓鼠试剂接种后确实生病。这一结果表明，星形胶质细胞特异性表达的HPrP也足以发生传输，但效率远低于如果表达仅限于神经元。此外，该结果证明了在表达MoPrP和HPrP的小鼠中MoPrP和HPrP之间的非常强的干扰。因为TSE疾病被认为是经口传播的，我们还用仓鼠瘙痒症剂经口和腹膜内接种NSE/HPrP Tg小鼠和另一个命名为Tg 7的Tg系，其中HPrP在多个组织中表达。大多数通过这些途径接种的小鼠如果同时表达小鼠和HPrP则存活。这些结果表明，TSE疾病的治疗干预可以基于这些干预机制。我们还发现，仓鼠瘙痒症病原体能够在脑内接种后的小鼠中持续存在，持续小鼠的正常寿命和临床瘙痒症的发病率（小鼠以前被认为对仓鼠瘙痒症病原体完全耐药）。这种持久性依赖于PrP的存在。我们确定了仓鼠试剂从小鼠中清除的动力学，并表明尽管大多数试剂被迅速消除，但持续存在的试剂最终可以适应新的物种，但需要盲传至第二个受体组。从第二次传代小鼠获得的脑和脾匀浆在仓鼠和小鼠（第三次传代）中引起临床疾病。在第四次传代时，各种菌株进化出各自的特征表型。因此，在被认为具有耐药性的种属中，仓鼠病原体持续存在至少4次连续传代。这一结果与BSE源自绵羊的理论一致，但表明牛中的疾病在牛中发生超过一次主要传代后才表现为临床疾病。持久性和最终适应抗性物种表明，类似的情况可能发生在其他物种组合。在美国，人们担心鹿和麋鹿的慢性消耗性疾病可能传染给其他物种，包括人类，尽管宿主范围尚未完全划定。我们加大了在慢性消耗病方面的努力，并已开始进行研究，以更准确地确定慢性消耗病的发病机制，包括各种组织的重要性，关于更好的诊断测试的可能性，并调查传播给人类的可能性。

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 occurred as the result of cattle being fed bone meal and protein supplements contaminated with tissue 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 people. Which TSE diseases will transmit to which other species 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 was able to persist in mice following intracerebral inoculation for the normal lifespan of the mouse and in the abscence of clinical scrapie ( mice had previously been considered totally resistant to hamster scrapie agent). This persistence was dependent on the presence of PrP. We determined the kinetics of hamster agent clearance from mice and showed that although most agent is rapidly eliminated that which persists can eventually adapt to the new species but required blind passage to a second recipient group. Brain and spleen homogenates obtained from the second pass mice caused clinical disease in hamsters as well as mice(3rd pass). On fourth passage various strains had evolved with their own characteristic phenotypes. Thus, hamster agent persisted through at least four serial passages in a species thought to be resistant. This result is consistent with the theory that BSE was derived from sheep but suggests that the disease in cattle was not manifest as clinical disease until more than a primary passage 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 have increased our effort with regard to CWD and have initiated studies to determine more precisely the pathogenesis of CWD including the importance of various tissues, possibilities regarding better diagnostic tests and investigating the potential for transmission to humans.