A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice

The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.

在绵羊中发现的数株自然羊瘙痒病分离株的蛋白酶抗性朊蛋白（PrPres），与源自英国自然羊瘙痒病病例的实验分离株CH1641特征相似，且此类分离株与绵羊源牛海绵状脑病（BSE）存在部分分子层面的相似性。近期在牛群中发现的非典型牛海绵状脑病毒株——L型BSE（或称BASE），提示该型BSE也有可能传播至绵羊体内。本研究通过蛋白质免疫印迹（Western Blot）技术，分析了4株“CH1641样”自然羊瘙痒病分离株在绵羊转基因模型（TgOvPrP4）中传代后的PrPres分子特征，以探究“CH1641样”分离株是否与L型BSE存在关联。研究发现，相较于经典型BSE，此类分离株的双糖基化PrPres含量更低，但其常规PrPres形式（PrPres #1）的糖型比例与表观分子量，均与L型BSE相近。但“CH1641样”分离株与L型及经典型BSE均存在差异：其可产生大量经羧基端截短的PrPres产物（PrPres #2），该产物可被羧基端特异性抗体SAF84识别。对PrPres #1与PrPres #2进行差异化免疫沉淀后，可实现PrPres #2的富集，并证实存在单糖基化与双糖基化的PrPres产物。在TgOvPrP4转基因小鼠中，数株实验性羊瘙痒病毒株（SSBP1、79A、Chandler、C506M3）无法产生PrPres #2；但87V羊瘙痒病毒株可检测到该产物，此外5株与CH1641分子特征存在差异的自然羊瘙痒病分离株中，也均以较低且波动的比例检出了PrPres #2。PrPres #2的鉴定为朊病毒毒株的分子鉴别提供了一种新方法，同时也证实了“CH1641样”绵羊瘙痒病与在绵羊转基因小鼠模型中传代的牛L型BSE之间存在差异。