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J. Biol. Chem., Vol. 282, Issue 41, 30022-30028, October 12, 2007

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Cross-sequence Transmission of Sporadic Creutzfeldt-Jakob Disease Creates a New Prion Strain^*

Atsushi Kobayashi, Masahiro Asano, Shirou Mohri, and Tetsuyuki Kitamoto¹

From the Division of CJD Science and Technology, Department of Prion Research, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan and Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan

Received for publication, June 5, 2007 , and in revised form, August 8, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of abnormal isoform of PrP (PrP^Sc) are major determinants of the clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Here we found that the transmission of sCJD prions from a patient with valine homozygosity (129V/V) and type 2 PrP^Sc (sCJD-VV2 prions) to mice expressing human PrP with methionine homozygosity (129M/M) generated unusual PrP^Sc intermediate in size between type 1 and type 2. The intermediate type PrP^Sc was seen in all examined dura mater graft-associated CJD cases with 129M/M and plaque-type PrP deposits (p-dCJD). p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology, and the identical type of PrP^Sc when inoculated into PrP-humanized mice with 129M/M or 129V/V. These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Creutzfeldt-Jakob disease (CJD),² kuru, scrapie, and bovine spongiform encephalopathy are lethal transmissible neurodegenerative diseases caused by an abnormal isoform of prion protein (PrP^Sc), which is converted from the normal cellular isoform (PrP^C) (1). The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of PrP^Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) (2–5). Type 1 and type 2 PrP^Sc are distinguishable according to the size of the proteinase K-resistant core of PrP^Sc (PrP^res) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (2, 3). The genotype at codon 129 also influences the susceptibility to variant CJD (vCJD), iatrogenic CJD, and kuru (6–11). A transmission study using transgenic mice expressing human PrP revealed that the congruency of the genotype at codon 129 between the inoculum and the inoculated transgenic mice determines the susceptibility to sCJD prions (12). Transmission of sCJD prions to mice with an incongruent genotype (referred to as cross-sequence transmission) results in a relatively long incubation period.

The potential for cross-sequence transmission should be considered in the iatrogenic transmission of CJD via cadaveric pituitary hormones, dura mater and corneal grafts, or contaminated neurosurgical instruments. More than half of the reported cases of dura mater graft-associated CJD (dCJD) occurred in Japan, where 123 cases have been recognized as of February 2006 (13–16). The dural grafts used in Japan were manufactured by German companies (13–15). In Europe, 28.4% of sCJD patients are valine homozygotes (129V/V) or methionine/valine heterozygotes at codon 129 (129M/V) (5). Meanwhile, the population data show a high prevalence (91.6%) of methionine homozygosity (129M/M) in Japanese people (17). These data raise the possibility that part of the Japanese dCJD cases might have been caused by cross-sequence transmission of sCJD prions. In fact, there are two distinct phenotypes in dCJD, with the majority represented by a non-plaque type of dCJD (np-dCJD) and the minority by a plaque-type dCJD (p-dCJD) (18–21). The clinicopathological features of np-dCJD are similar to those of sCJD with 129M/M and type 1 PrP^Sc (sCJD-MM1) (14). In contrast, p-dCJD cases show unique features characterized by (i) the absence or late occurrence of myoclonus and periodic synchronous discharges on electroencephalogram; (ii) a long incubation period after grafting and a clinical course of long duration, and (iii) plaque-type PrP deposits in the brain (18–25). Although we have classified p-dCJD cases into MM1, the clinicopathological features of p-dCJD are quite different from those of sCJD-MM1 or np-dCJD (18–21). The reason for the existence of two distinct phenotypes in dCJD has remained elusive.

To identify the origin of p-dCJD, we inoculated sCJD prions into mice expressing human PrP with either 129M/M or 129V/V. Here we report that cross-sequence transmission of sCJD prions from a patient with 129V/V and type 2 PrP^Sc (sCJD-VV2 prions) caused phenotypes similar to those of p-dCJD.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production of Knock-in Mice and Transgenic Mice—The production of knock-in mice expressing human PrP with 129M/M (Ki-Hu129M/M) and Ki-Hu129V/V mice has been reported previously (8). Knock-in mice expressing human PrP with 129M/M and four octapeptide repeats (Ki-Hu129M_4R/M_4R), transgenic mice expressing human PrP with 129M and four octapeptide repeats (Tg-Hu129M_4R), and Tg-Hu129M mice were generated as described (26, 27). To evaluate the effect of overexpression of the PrP gene, Ki-Hu129M_4R/M_4R, Ki-Hu129M/M, and Ki-Hu129V/V mice were crossed with Tg-Hu129M_4R, Tg-Hu129M, or Tg-Hu129V mice (8). The expression levels of human PrP in the brains from Tg+Ki-Hu129M_4R/M_4R, Tg+Ki-Hu129M/M, and Tg+Ki-Hu129V/V mice were 9.8x, 1.2x, and 2.1x, respectively, the levels observed in Ki-Hu129M/M or Ki-Hu129V/V mice.

Human Brain Inocula—Brain tissues were obtained at autopsy from CJD patients after receiving informed consent for research use. The diagnosis of CJD and the type of PrP^Sc were confirmed by neuropathological examination, PrP^Sc immunohistochemistry, and Western blotting as described (28, 29). The genotype and the absence of mutations in the coding region of the PrP gene were determined by sequence analysis (30).

Transmission Experiments—Human brain homogenates (10%) and mouse brain homogenates (10%) were prepared as described (27). Transmission studies were performed using 20 µl of the homogenates for intracerebral inoculation or 50 µl for intraperitoneal inoculation (8, 29). Intracerebrally inoculated mice were sacrificed after the onset of disease, and their brains were immediately frozen or fixed in 10% buffered formalin. Intraperitoneally inoculated mice were sacrificed at 75 days post-inoculation, and their spleens were immediately frozen or formalin-fixed.

Immunohistochemistry—Formalin-fixed mouse brains and spleens were treated with 60% formic acid for 1 h to inactivate the infectivity and embedded in paraffin. Tissue sections were pretreated by hydrolytic autoclaving before PrP immunohistochemistry (28). The PrP-N antiserum was used as the primary antibody (31). Goat anti-rabbit immunoglobulins polyclonal antibody labeled with the peroxidase-conjugated dextran polymer, EnVision+ (DakoCytomation) were used as the secondary antibody.

Western Blotting—PrP^Sc was extracted from human brains or mouse brains and spleens with collagenase treatment as described (32) with some modifications. For deglycosylation of PrP, samples were treated with PNGase F (New England Biolabs). Samples were subjected to 13.5% SDS-PAGE and Western blotting as described (8). The 3F4 monoclonal antibody (Signet Laboratories) and the ChW antiserum (8) were used as the primary antibodies. Anti-mouse EnVision+ and anti-rabbit EnVision+ were used as the secondary antibodies.

Statistical Analysis—Incubation times are expressed as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transmission of sCJD Prions to PrP-humanized Mice with 129M/M or 129V/V—To investigate whether strain-dependent traits of sCJD prions can be inherited through cross-sequence transmission, we performed intracerebral inoculation of a brain homongenate from a sCJD-MM1 patient (H3) or that from a sCJD-VV2 patient (AK) into PrP humanized mice with the 129M/M or 129V/V genotype. For sCJD-MM1 prions, the incubation times of Ki-Hu129V/V mice were 542 and 648 days (number of diseased animals/number of inoculated animals = 2/5), which were longer than those of Ki-Hu129M/M mice (467 ± 24 days, 8/8) (Table 1). For sCJD-VV2 prions, the mean incubation time of Tg+Ki-Hu129M/M mice was 723 ± 79 days (4/4), which was longer than that of Ki-Hu129V/V mice (312 ± 7 days, 4/4). Immunohistochemical analysis showed diffuse synaptic-type PrP deposits in the brains from both Tg+Ki-Hu129M/M and Tg+Ki-Hu129V/V mice inoculated with sCJD-MM1 prions (Fig. 1A). In contrast, Tg+Ki-Hu129V/V mice inoculated with sCJD-VV2 prions showed small plaque-type PrP deposits in the cerebral white matter and granular to diffuse synaptic-type deposits in the gray matter. The plaque-type PrP deposits were more prominent in the brains from Tg+Ki-Hu129M/M mice inoculated with sCJD-VV2 prions, in which larger plaque-type PrP deposits spread throughout the cerebral gray matter and thalamus rather than the white matter. Western blot analysis revealed that the size of PrP^res in the brains was maintained after cross-sequence transmission of sCJD-MM1 prions but not sCJD-VV2 prions (Fig. 1, B and C). Tg+Ki-Hu129V/V mice inoculated with sCJD-MM1 prions produced type 1 PrP^res (hereafter denoted as VV[MM1]1 PrP^res:host genotype[inoculated prions]type of generated PrP^res), which were identical in size to MM[MM1]1 PrP^res from Tg+Ki-Hu129M/M mice. However, Tg+Ki-Hu129M/M mice inoculated with sCJD-VV2 prions produced unusual PrP^res that were larger than VV[VV2]2 PrP^res from Tg+Ki-Hu129V/V mice but smaller than MM[MM1]1 or VV[MM1]1 PrP^res. We designated this intermediate-sized PrP^res as MM[VV2]2^Sh+, PrP^res with an upward size shift (Sh+) from the inoculated type 2 template. The shift in the size of PrP^res and the prominent plaque formation in Tg+Ki-Hu129M/M mice indicated that the strain-dependent traits of sCJD-VV2 prions were modified through cross-sequence transmission.

Transmission of p-dCJD Prions to PrP-humanized Mice with 129M/M or 129V/V—To investigate the transmissibility of p-dCJD prions, we performed intracerebral inoculation of brain homogenates from p-dCJD patients (KR, KD, TV) into PrP-humanized mice with the 129M/M or 129V/V genotype. We had already established Ki-Hu129M_4R/M_4R mice expressing human PrP with 129M/M and four octapeptide repeats before we produced Ki-Hu129M/M mice expressing human PrP with five octapeptide repeats. The four or five octapeptide repeats of human PrP are polymorphisms unassociated with CJD (33). For sCJD-MM1 prions, these Ki-Hu129M_4R/M_4R mice showed long incubation times of >600 days in our preliminary experiment (data not shown). Therefore, they were crossed with Tg-Hu129M_4R mice to overexpress the human PrP gene. Because these Tg+Ki-Hu129M_4R/M_4R mice were highly susceptible to sCJD-MM1 prions (175 ± 4 days, 9/9) (Table 1), we used them as PrP-humanized mice with the 129M/M genotype in this experiment at first. All of Tg+Ki-Hu129M_4R/M_4R and Ki-Hu129V/V mice inoculated with p-dCJD prions developed disease (Table 2). The mean incubation times of Tg+Ki-Hu129M_4R/M_4R mice were 398 ± 10, 420 ± 10, and 584 ± 65 days. Though the expression level of human PrP in Tg+Ki-Hu129M_4R/M_4R mice was 9.8-fold higher than that of Ki-Hu129V/V mice, the mean incubation times of Tg+Ki-Hu129M_4R/M_4R mice were longer than those of Ki-Hu129V/V mice (259 ± 6 and 304 ± 13 days). For np-dCJD, the mean incubation times of Tg+Ki-Hu129M_4R/M_4R mice were 161 ± 5 (5/5) and 208 ± 2 days (5/5). Immunohistochemical analysis of the brains from Tg+Ki-Hu129M_4R/M_4R mice inoculated with p-dCJD prions showed a few plaque-type PrP deposits similar to those in Tg+Ki-Hu129M_4R/M_4R mice inoculated with sCJD-VV2 prions (Fig. 3A). Ki-Hu129V/V mice inoculated with p-dCJD prions showed small plaque-type PrP deposits in the cerebral white matter and granular to diffuse synaptic-type deposits in the gray matter similar to those in Ki-Hu129V/V mice inoculated with sCJD-VV2 prions. There was no plaque-type PrP deposits in the brains from Tg+Ki-Hu129M_4R/M_4R mice inoculated with np-dCJD prions (Fig. 3B). Western blot analysis revealed that Tg+Ki-Hu129M_4R/M_4R mice inoculated with p-dCJD prions produced the intermediate type PrP^res (M_4RM_4R[MMi]i PrP^res), which were identical in size to M_4RM_4R[VV2]2^Sh+ PrP^res (Fig. 3, C and D). Furthermore, Ki-Hu129V/V mice inoculated with p-dCJD prions produced type 2 PrP^res (VV[MMi]2 PrP^res), which were identical in size to VV[VV2]2 PrP^res. Thus, p-dCJD prions and sCJD-VV2 prions were similar in the transmissibility, the patterns of PrP deposition, and the size of PrP^res (the intermediate type in Tg+Ki-Hu129M_4R/M_4R or type 2 in Ki-Hu129V/V) in PrP-humanized mice.

View larger version (72K): [in this window] [in a new window]   FIGURE 1. Strain-dependent traits of sCJD-VV2 prions were modified through cross-sequence transmission. A, immunohistochemical analysis of the brains from Tg+Ki-Hu129M/M mice inoculated with sCJD-VV2 prions showed prominent plaque-type PrP deposits (arrowheads) throughout the cerebral gray matter and thalamus. In the brains from Tg+Ki-Hu129V/V mice inoculated with sCJD-VV2 prions, plaque-type PrP deposits were restricted to within the white matter. B, Western blot analysis of the brains from PrP-humanized mice after proteinase K digestion. C, proteinase K-digested samples were treated with PNGase F for deglycosylation of PrPres. Tg+Ki-Hu129M/M mice inoculated with sCJD-VV2 prions produced unusual PrPres intermediate in size between type 1 and type 2 (designated as MM[VV2]2Sh+ PrPres:host genotype[inoculated prions]type of generated PrPres).

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Intermediate type PrPres (designated as MMi PrPres) was a common form detected in human p-dCJD cases. A, Western blot analysis of the brains from dCJD patients or those from sCJD-MM1 patients after proteinase K digestion. B, for deglycosylation of PrPres, proteinase K-digested samples were treated with PNGase F. PrPres in the brains from p-dCJD patients were smaller than MM1 PrPres from sCJD-MM1 or np-dCJD cases.

We inoculated intracerebrally a brain homogenate from a p-dCJD patient (KD) into Ki-Hu129M/M mice besides Tg+Ki-Hu129M_4R/M_4R mice. The mean incubation time of these Ki-Hu129M/M mice was 500 ± 59 days (4/4). These mice produced MM[MMi]i PrP^res identical in size to M_4RM_4R[MMi]i PrP^res from Tg+Ki-Hu129M_4R/M_4R mice (Fig. 3F).

Intraperitoneal Transmission of p-dCJD Prions or sCJD-VV2 Prions to PrP-humanized Mice with 129M/M or 129V/V—To confirm the similarity in the transmissibility between p-dCJD prions and sCJD-VV2 prions, we performed intraperitoneal inoculation of the brain homogenate from a p-dCJD patient (KD) or that from a sCJD-VV2 patient (AK) into PrP-humanized mice as described previously (27). Immunohistochemical analysis of the spleens at 75 days post-inoculation revealed that Ki-Hu129V/V mice were more susceptible to p-dCJD prions than Ki-Hu129M/M mice (Fig. 4A). Similarly, Ki-Hu129V/V mice were more susceptible to sCJD-VV2 prions than Ki-Hu129M/M mice. In contrast, Ki-Hu129V/V mice intraperitoneally inoculated with sCJD-MM1 prions showed no obvious PrP deposition in the spleen (0/9). Therefore, we confirmed that p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility to PrP-humanized mice even in peripheral infection. In addition, Ki-Hu129M/M mice intraperitoneally inoculated with sCJD-VV2 prions produced MM[VV2]2^Sh+ PrP^res in the spleen (Fig. 4B). Meanwhile, Ki-Hu129V/V mice intraperitoneally inoculated with sCJD-VV2 prions produced VV[VV2]2 PrP^res. Thus, we confirmed the shift of the PrP^res size also in the spleens.

Because cross-sequence transmission of sCJD-VV2 prions to Tg+Ki-Hu129M/M mice caused phenotypes similar to those of p-dCJD, we also examined the transmissibility of Tg+Ki-Hu129M/M mouse-passaged sCJD-VV2 prions (designated as MM[VV2]2^Sh+ prions). We performed intraperitoneal inoculation of the brain homogenate from a Tg+Ki-Hu129M/M mouse that succumbed to sCJD-VV2 prions into PrP-humanized mice. All (11/11) of Ki-Hu129V/V mice inoculated with MM[VV2]2^Sh+ prions showed PrP deposition in the spleen despite cross-sequence transmission. Western blot analysis of the spleens revealed that Ki-Hu129M/M mice produced type 2^Sh+ PrP^res (MM[MM[VV2]2^Sh+]2^Sh+ PrP^res) identical in size to MM[VV2]2^Sh+ PrP^res. Meanwhile, Ki-Hu129V/V mice produced type 2 PrP^res (VV[MM[VV2]2^Sh+]2 PrP^res) identical in size to VV[VV2]2 PrP^res (Fig. 4B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Our data comprised of four major findings. First, transmission of sCJD-VV2 prions to Tg+Ki-Hu129M/M mice generated unusual PrP^res (MM[VV2]2^Sh+ PrP^res) intermediate in size between type 1 and type 2. Second, the intermediate type MMi PrP^res was seen in all examined p-dCJD cases. Third, Ki-Hu129V/V mice inoculated with p-dCJD prions showed short incubation times and the accumulation of type 2 PrP^res with a downward size shift from the intermediate type template. Finally, Ki-Hu129V/V mice intraperitoneally inoculated with MM[VV2]2^Sh+ prions showed high susceptibility and the accumulation of type 2 PrP^res with a downward size shift from type 2^Sh+ template. These findings suggest that p-dCJD could be caused by transmission of sCJD-VV2 prions to individuals with the 129M/M genotype (cross-sequence transmission).

View larger version (57K): [in this window] [in a new window]   FIGURE 3. p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology and identically sized PrPres in PrP-humanized mice. A, immunohistochemical analysis of the brains from Tg+Ki-Hu129M4R/M4R mice inoculated with p-dCJD prions or sCJD-VV2 prions showed a few plaque-type PrP deposits (arrowheads). Ki-Hu129V/V mice inoculated with p-dCJD prions or sCJD-VV2 prions showed small plaque-type PrP deposits in the cerebral white matter and synaptic-type deposits in the gray matter. B, diffuse synaptic-type PrP deposits in the brains from Tg+Ki-Hu129M4R/M4R mice inoculated with np-dCJD prions or sCJD-MM1 prions. There was no plaque-type PrP deposits. C, Western blot analysis of the brains from PrP-humanized mice after proteinase K digestion. D, for deglycosylation of PrPres, proteinase K-digested samples were treated with PNGase F. Tg+Ki-Hu129M4R/M4R mice inoculated with p-dCJD prions or sCJD-VV2 prions produced the intermediate type PrPres. Meanwhile, Ki-Hu129V/V mice inoculated with p-dCJD prions or sCJD-VV2 prions produced type 2 PrPres. E, Tg+Ki-Hu129M4R/M4R mice inoculated with sCJD-MM1 prions produced M4R/M4R[MM1]1 PrPres identical in size to MM[MM1]1 PrPres from Ki-Hu129M/M mice. Tg+Ki-Hu129M4R/M4R mice inoculated with sCJD-VV2 prions produced M4R/M4R[VV2]2Sh+ PrPres identical in size to MM[VV2]2Sh+ PrPres from Tg+Ki-Hu129M/M mice. F, Ki-Hu129M/M mice inoculated with p-dCJD prions produced MM[MMi]i PrPres identical in size to M4R/M4R[MMi]i PrPres from Tg+Ki-Hu129M4R/M4R mice.

sCJD cases with the 129M/V genotype and type 2 PrP^Sc (sCJD-MV2) and sCJD-VV2 cases account for 25% of the total sCJD cases and show the characteristic neuropathological changes highlighted by plaque-type PrP deposits (5). To date, some similarities among sCJD-MV2, sCJD-VV2, and p-dCJD have been described in clinicopathological, biochemical, and transmission studies (18–21, 29). Although the results of the present study show that cross-sequence transmission of sCJD-VV2 prions can cause phenotypes similar to those of p-dCJD, further studies are needed to clarify whether sCJD-MV2 prions can cause a p-dCJD like phenotype in humanized mice with 129M/M.

Besides p-dCJD, a few iatrogenic CJD cases with the 129M/M genotype and plaque-type PrP deposits in the brain have been reported in human growth hormone-related CJD (34, 35). The present results lead us to surmise that the human growth hormone-related CJD cases with 129M/M and plaque-type PrP deposits might be caused by the cross-sequence transmission of sCJD-VV2 prions.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Intraperitoneal transmission of p-dCJD prions, sCJD-VV2 prions, or Tg+Ki-Hu129M/M mouse-passaged sCJD-VV2 prions (designated as MM[VV2]2Sh+ prions) to PrP-humanized mice. A, Ki-Hu129V/V mice were highly susceptible to p-dCJD prions and sCJD-VV2 prions even in peripheral infection. B, Western blot analysis of the spleens after proteinase K digestion and PNGase F treatment. Ki-Hu129M/M mice intraperitoneally inoculated with sCJD-VV2 prions produced MM[VV2]2Sh+ PrPres identical in size to those seen in the brain after intracerebral inoculation. Ki-Hu129M/M mice inoculated with MM[VV2]2Sh+ prions produced type 2Sh+ PrPres identical in size to MM[VV2]2Sh+ PrPres. Meanwhile, Ki-Hu129V/V mice inoculated with MM[VV2]2Sh+ prions produced type 2 PrPres.

The above concept is supported by the "traceback" phenomenon (8), e.g. knock-in mice and transgenic mice expressing bovine PrP are highly susceptible to vCJD prions as well as bovine spongiform encephalopathy prions (8, 39). Consistent with a report using transgenic mice expressing human PrP with 129M or 129V (12), the humanized mice with 129V/V in our study were more susceptible to sCJD-VV2 prions than the humanized mice with 129M/M. Furthermore, the humanized mice with 129V/V showed high susceptibility to MM[VV2]2^Sh+ prions and p-dCJD prions despite cross-sequence transmission. These phenomena can be explained as follows. Because MM[VV2]2^Sh+ prions and p-dCJD prions retained the memory of the parental sCJD-VV2 prions, the humanized mice with 129V/V were highly susceptible to these prions as well as sCJD-VV2 prions. Our results demonstrate that traceback studies can be a powerful tool to identify the origin of prions.

In this study, the strain-dependent traits of sCJD-MM1 prions were inherited through cross-sequence transmission without any modification. The humanized mice with 129V/V produced type 1 PrP^res after inoculation with sCJD-MM1 prions. Because sCJD-VV1 cases are extremely rare (at most 1–2% of the total number of sCJD cases) and characterized by early onset (mean age at onset, 39.3 years) (5), our results raise the possibility that CJD cases classified as VV1 may include cases caused by iatrogenic transmission of sCJD-MM1 prions or food-borne infection by type 1 prions from animals, e.g. chronic wasting disease prions in cervid. In fact, two CJD-VV1 patients who hunted deer or consumed venison have been reported (40, 41). The results of the present study emphasize the need for traceback studies and careful re-examination of the biochemical properties of sCJD-VV1 prions.

In conclusion, cross-sequence transmission of sCJD-VV2 prions generates a new prion strain with altered conformational properties and disease phenotypes as p-dCJD prions. Furthermore, the newly generated prions have unique transmissibility including the traceback phenomenon. In the future, if atypical prion strains emerge through cross-sequence transmission, especially from animals, traceback studies will enable us to identify the origin of the prions.

	FOOTNOTES

 
^* This work was supported by the program for Promotion of Fundamental Studies in Health Sciences of Japanese National Institute of Biomedical Innovation (to S. M. and T. K.), a grant from the Ministry of Health, Labor and Welfare of Japan (to A. K., S. M., and T. K.), and a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A. K. and T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Div. of CJD Science and Technology, Dept. of Prion Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Tel.: 81-22-717-8143; Fax: 81-22-717-8148; E-mail: kitamoto{at}mail.tains.tohoku.ac.jp.

² The abbreviations used are: PrP, prion protein; PrP^C, normal cellular isoform of PrP; PrP^Sc, abnormal isoform of PrP; PrP^res, proteinase K-resistant core of PrP^Sc; 129M/M, methionine homozygosity at codon 129 of the human PrP gene; 129V/V, valine homozygosity at codon 129 of the human PrP gene; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; p-dCJD, plaque-type dura mater graft-associated CJD; np-dCJD, non-plaque type dura mater graft-associated CJD; vCJD, variant CJD; dCJD, dura mater graft-associated CJD.

	ACKNOWLEDGMENTS

 
We thank H. Kudo and K. Abe for technical assistance and R.-W. Shin and B. Bell for critical review of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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