Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Figures & data

Figure 1 Neuropathology and PrPcore characteristics of L-BSE-affected TgHaNSE mice. (A) hematoxylin and eosin staining of the corpus callosum of mice. (B) PrP^Sc deposition was detected in the semiserial sections. PrP-plaque is indicated by an arrow. PrP was detected by mAb SAF-84. Scale bars: 200 mm. (C) PrP^Sc in L-BSE affected TgHaNSE mice was detected by western blotting. Lane 1: C-BSE (cattle), lane 2: L-BSE (cattle), lane 3: C-BSE affected hamster, lane 4: L-BSE affected hamster, lanes 5 and 6: L-BSE affected TgHaNSE, lane 7: scrapie Obihiro-affected mouse. PrP was detected by mAb T2.

Figure 2 Neuropathological lesion profiling and PrP^Sc deposition in hamsters. (A) Vacuolar lesion profiles in Syrian hamster brains as observed for scrapie strains Sc237, C-BSE-affected hamsters (C-BSE) and L-BSE-affected hamsters (L-BSE). Gray matter scoring areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, medial thalamus; 6, hippocampus; 7, septum; 8, posterior cerebral cortex; 9, anterior cerebral cortex. White matter scoring areas: 1*, cerebellar white matter; 2*, midbrain white matter; 3*, cerebral peduncle. Mean (standard deviation) (n = 4). (B) PrP^Sc deposition in the brains of hamsters affected with C-BSE (second passage), L-BSE (second passage) and Sc237 (serial passage). MAb SAF84 was used for immunostaining. Upper, cerebral cortex; lower, cerebellum.

Figure 3 Western blotting analysis of PrPcore from C-BSE- and L-BSE-affected hamsters. (A) Lane 1: C-BSE-affected cattle (natural case); lane 2: L-BSE-affected cattle (natural case); lanes 3 and 6: Sc237-affected hamsters; lanes 4 and 7: C-BSE-affected hamsters; lanes 5 and 8: L-BSE-affected hamsters; lanes 6–8: PNGaseF treatment. (B) The relative amount (%) of di-, mono- and non-glycosylated PrPcore. The results are the mean (standard deviation) of three experiments. Bar diagram: di- (black), mono-(grey) and nonglycosylated form (white).

Figure 4 Relative PK resistance of PrPcore in prion-affected hamsters. (A) Western blot results. Lanes 1–4: hamster-adapted C-BSE; lanes 5–8: L-BSE-affected hamster. Lane 9: mouse scrapie prion. The samples were treated with 40 (lanes 1 and 5), 100 (lanes 2 and 6), 500 (lanes 3 and 7) and 1,000 (lanes 4 and 8) µg/ml of PK at 37°C for 1 h. PrPcore was detected with mAb 6H4. Molecular markers are shown on the left (kDa). (B) Relative amount (%) of PrPcore after different PK concentration were indicated. Black circle: C-BSE affected hamster, black square: L-BSE affected hamster, white circle: C-BSE affected cattle, white square: L-BSE affected cattle. Cattle results are obtained from previous study in reference 11.

Table 1 Transmissibility of L-BSE in mice

Mice	Number of mice affected/inoculated	Incubation period (days)
Mouse	0/5	>700*
tga20	0/5	>668*
MHM2	0/5	>755*
MH2M	0/5	>853*
TgHaNSE	2/3	567†, 853†

The brain homogenate of L-BSE was intracerebrally inoculated into mice.

* Mice showed no clinical signs, and tested negative for PrP^Sc in the brain.

† PrP^Sc was present in the brain.

Table 2 Incubation period of hamsters inoculated with C-BSE and L-BSE

Inoculum	Passage numbers	Number of hamsters diseased/inoculated	Incubation period mean (SD) (days)
C-BSE	1st	0/5	>600*
Mouse passaged C-BSE†	1st	10/10	349.5 (6.6)
	2nd	10/10	267.3 (19.2)
	3rd	10/10	271.6 (16.2)
L-BSE	1st	3/4	576.8 (127.8)
	2nd	4/4	208 (15.5)

* Hamsters showed no clinical signs and were negative for PrP^Sc in the brain.

† After one passage of BSE from cattle in mice (incubation period, 408.6 (28.2) days) and subsequent transmission to TgHaNSE mice (incubation period, 153.1 (1.1) days).14 The brain of a diseased TgHaNSE mouse was inoculated in a hamster.

Table 3 Comparison of the PrP amino acid sequences in mouse and hamster

Species	Susceptibility				PrP amino acid*
	C-BSE†	L-BSE	109	112	139	155	170	203	205	215
Mouse	Sus‡	Res§	L	V	I	Y	S	V	M	V
MHM2	Sus†	Res	M	M	-¶	-	-	-	-	-
MH2M	Res†	Res	M	M	M	N	N	-	-	-
Hamster	Res†	Sus	M	M	M	N	N	I	I	T
Cattle#	Sus	Sus	M	-	-	N	-	I	-	I

* Residue numbers correspond to those in hamster PrP.

† Transmission results of C-BSE were reported previously.14

‡ Susceptible.

§ Resistant.

¶ Indicates the same amino acid sequence as mouse PrP.

# Transmission results of cattle were reported previously.8

Masujin K, Shu Y, Yamakawa Y, Hagiwara K, Sata T, Matsuura Y, et al. Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle. Prion 2008; 2:123 - 128

 PubMed Web of Science ®Google Scholar

Yokoyama T, Masujin K, Iwamaru Y, Imamura M, Mohri S. Alteration of the biological and biochemical characteristics of bovine spongiform encephalopathy prions during interspecies transmission in transgenic mice models. J Gen Virol 2009; 90:261 - 268

 Google Scholar

Fukuda S, Iwamaru Y, Imamura M, Masujin K, Shimizu Y, Matsuura Y, et al. Intraspecies transmission of L-type-like Bovine Spongiform Encephalopathy detected in Japan. Microbiol Immunol 2009; 53:704 - 707

 Google Scholar