Strain-Specific Barriers against Bovine Prions in Hamsters

Abstract

We investigated the susceptibilities of Syrian golden hamsters to transmissible spongiform encephalopathy agents from cattle. We report efficient transmission of the L-type atypical bovine spongiform encephalopathy (BSE) agent into hamsters. Importantly, hamsters were also susceptible to the transmissible mink encephalopathy agent from cattle, which has molecular features similar to those of the L-type BSE agent, as also shown in bovinized transgenic mice. In sharp contrast, hamsters could not be infected with classical or H-type BSE agents from cattle. However, previous adaptation of the classical BSE agent in wild-type mice led to efficient transmission. Thus, this study demonstrates the existence of distinct “strain barriers” upon the transmission of bovine prions in hamsters.

In recent years, bovine spongiform encephalopathy (BSE) cases with atypical molecular and/or neuropathological phenotypes have been reported in several European countries, in North America, and in Japan (12, 13, 17). Atypical BSE agents are currently classified into two types, L and H, according to the slightly lower (L) or higher (H) molecular mass of the protease-resistant prion protein (PrP^res) detected by Western blot analysis compared with the molecular mass of the classical (C-type) food-borne BSE agent. Given their low prevalence worldwide, such BSE cases have been assumed to represent sporadic forms of prion diseases.

Both of these atypical BSE agents have been experimentally challenged in different hosts, including cattle, sheep, monkeys, and wild-type or transgenic mice. Altogether, these experiments have demonstrated the infectious nature of such BSE cases and the existence of at least three distinct major prion strains in cattle. However, recent studies have indicated that the L-type BSE agent may acquire phenotypic features similar to those of the C-type BSE agent after inoculation into an ovine transgenic (tg338) mouse model or into inbred wild-type mice (4, 8). This supports the hypothesis that sporadic forms of BSE agents were possibly at the origin of the classical BSE epidemic in cattle.

The Syrian golden hamster has already proved to be a useful experimental model for studying the infectious agents involved in some transmissible spongiform encephalopathies (TSEs). The initial isolation of two biologically different prion strains, associated with distinct PrP properties, in transmissible mink encephalopathy (TME) was first achieved in hamsters (5). Surprisingly, bioassay studies in hamsters after challenge with the C-type BSE agent from cattle showed that it was inefficient (11, 21). This was attributed to the existence of a so-called “species barrier,” a concept based on the observation that the transmission of prion diseases between different species is typically far less efficient than within species (10).

In the present study, we investigated the susceptibilities of Syrian golden hamsters to TSE agents from cattle, including the three naturally occurring types of BSE agents (classical, H, and L types), as well as an original isolate of the TME agent that had been experimentally passaged in cattle (15). For comparison in a homologous model, we also challenged bovinized transgenic mice (BoPrP-Tg110) (9) with these four bovine isolates.

The bovine TSE isolates used as inoculums in this study have been described previously (1). They included (i) a classical BSE isolate (01-2281), injected either as a homogenate of the original bovine brain tissue or after a first passage in C57BL/6 mice (2), (ii) an H-type BSE isolate (03-2095), (iii) an L-type BSE isolate (02-2528), and (iv) the Stetsonville TME isolate experimentally passaged in cattle (15). PrP^res from these bovine inoculums has been characterized previously by Western blot analysis (1). This showed the original molecular characteristics as reported for BSE isolates (classical, H, and L types) from field cases (13). In addition, we injected into hamsters the sheep scrapie brain pool 1 (SSBP/1) experimental scrapie isolate passaged in ovine transgenic mice (TgOvPrP4) (3).

For each TSE source, four or five (Table 1) male Syrian golden hamsters (Charles River, Germany) were inoculated intracerebrally with 30 μl of a 10% (wt/vol) brain homogenate in 5% glucose and were monitored until the end of life for the presence of any signs of distress or possible clinical signs indicative of prion disease. For bovinized transgenic mouse experiments, groups of 9 to 13 mice (Table 1) were inoculated intracerebrally with 20 μl of the same bovine brain homogenates. The resulting survival data are summarized in Table 1. PrP^res extractions using ultracentrifugation and Western blot analyses have been described previously (1). PrP^res was detected by using SAF84 or 12B2 monoclonal antibodies, against the hamster 164-RPVDQY-169 and 89-WGQGG-93PrP sequences, respectively.

TABLE 1.

TSE sources injected into Syrian golden hamsters and bovinized transgenic mice (BoPrP-Tg110) and the resulting transmission data

TSE inoculum	Hamsters		BoPrP-Tg110 mice
	Mean survival time (d.p.i. ± SD)	No. with PrPres/total no.	Mean survival time (d.p.i. ± SD)	No. with PrPres/total no.
From cattle
C-type BSE agent	777 ± 142	0/5	279 ± 17	13/13
H-type BSE agent	794 ± 54	0/4	341 ± 81	10/10
L-type BSE agent	622 ± 64	4/5	225 ± 35	11/11
TME agent passaged in cattle	764 ± 42	4/5	191 ± 31	9/9
Passaged in mice
Scrapie SSBP/1a	489 ± 79	4/4
C-type BSE agentb	386 ± 12	5/5

^aOne passage in transgenic mice expressing ovine prion protein (TgOvPrP4).

^bOne passage in wild-type mice (C57BL/6).

Primary transmission of the classical and H-type BSE agents from cattle into Syrian hamsters was inefficient, whereas both the L-type BSE agent and the TME agent passaged in cattle were efficiently transmitted, as assessed by PrP^res accumulation in the brain of terminally affected animals (Table 1) from 555 and 710 days postinoculation (d.p.i.), respectively. None of the nine hamsters inoculated with the bovine classical or H-type BSE agent exhibited detectable PrP^res up to 937 and 870 d.p.i., respectively. Although significantly shorter for the L-type BSE agent (mean survival of 622 d.p.i.) (P < 0.005), survival periods did not differ significantly between hamsters inoculated with other bovine TSE agents (mean survival periods of 764, 777, and 794 d.p.i.) (P > 0.3), probably reflecting the “species barrier” effect during primary transmissions. Regarding classical and H-type BSE agents, two and three hamsters, respectively, were found dead, the others being sacrificed for welfare reasons toward the maximum life span of the animals. The lack of transmissibility of the classical and H-type BSE agents in hamsters is not likely attributable to major differences in the infectious titers of the inoculums, as they were readily transmitted to bovinized transgenic mice (Table 1), with 100% attack rates, as previously described (4, 7, 16). Importantly, in the cases of the L-type BSE agent and the TME agent passaged in cattle, all 10 hamsters were sacrificed because of clinical signs that included prostration, hunched posture, clasping, and difficulty or incapacity in standing up. Hamsters inoculated with mouse-passaged TSE agents, which showed a 100% attack rate, exhibited shorter survival periods than did hamsters inoculated with bovine isolates, and obvious signs of aggressiveness were specifically identified in the case of the classical BSE agent transmitted from infected C57BL/6 mice.

Western blot molecular typing of the unglycosylated PrP^res band in terminally affected hamsters using the SAF84 antibody (Fig. 1 A) showed, in comparison with the highest band in TgOvPrP4 mouse-passaged SSBP/1 (∼19.5 kDa), (i) a slightly although not significantly lower molecular mass (0.20 kDa; P = 0.1) in the C57BL/6-passaged classical BSE agent and (ii) a significantly lower molecular mass (0.78 kDa; P < 0.001) that was indistinguishable (P = 1) in both the L-type BSE agent and the TME agent passaged in cattle. These observations were confirmed after protein deglycosylation (Fig. 1B). Differences were also readily apparent with the N-terminal 12B2 antibody, which showed strong PrP^res labeling in SSBP/1 from TgOvPrP4 mice, reduced labeling in the classical BSE agent from C57BL/6 mice, and no detectable signal in either the L-type BSE agent or the TME agent passaged in cattle (Fig. 1C). Molecular typing in bovinized transgenic mice (Fig. 1D to F) showed (i) the maintenance of the original bovine PrP^res patterns in the three BSE agent types (4, 7, 16), notably for the slightly lower molecular mass of unglycosylated PrP^res in the L-type BSE agent in comparison to that of the classical BSE agent, and (ii) indistinguishable molecular characteristics in the L-type BSE agent and the TME agent passaged in cattle. The biochemical features in the H-type BSE agent also included the characteristic ∼14-kDa C-terminal PrP^res fragment, as previously described (2, 6, 16).

FIG. 1.

Western blot molecular typing of PrP^res in the brains of terminally affected hamsters and BoPrP-Tg110 mice. (A to C) PrP^res in hamsters. TgOvPrP4 ovine transgenic mouse-passaged SSBP/1 (lane 1), C57BL/6-passaged classical BSE agent (lanes 2, 4, and 6), L-type BSE agent (lane 3), and TME agent passaged in cattle (lane 5) are compared. (D to F) PrP^res in BoPrP-Tg110 mice. Classical BSE agent (lane 7), H-type BSE agent (lane 8), L-type BSE agent (lane 9), and TME agent passaged in cattle (lane 10) are compared. PrP^res was detected either by using the monoclonal antibody SAF84 before (A, D) or after (B, E) deglycosylation or by using the monoclonal antibody 12B2 (C, F). Equivalent brain quantities were loaded in the experiments shown in panels A and C and in panels D and F, respectively. Bars to the left of the panels indicate the 29.0-, 20.1-, or 14.3-kDa (only in panels D and E) marker positions.

The analyses of PrP^res glycoform ratios in the brains of terminally affected hamsters (Fig. 2 A) showed that the L-type BSE agent and the TME agent passaged in cattle were indistinguishable (P = 1) but also did not differ significantly from the C57BL/6 mouse-passaged classical BSE agent (P = 0.1 and P = 0.2, respectively). In SSBP/1-infected hamsters, however, the PrP^res glycoform ratios were significantly distinct from those of the three other TSE agents (P < 0.001). Glycoform analysis of PrP^res from bovinized transgenic mice (Fig. 2B) essentially showed similar and balanced proportions of di- and monoglycosylated PrP^res in the L-type BSE agent and the TME agent passaged in cattle, which was clearly different from the results for the classical and H-type BSE agents.

FIG. 2.

Proportions of PrP^res glycoforms in the brains of terminally affected hamsters and BoPrP-Tg110 mice. Diglycosylated (dark gray), monoglycosylated (light gray), and unglycosylated (black) PrP^res glycoforms were quantified using monoclonal antibody SAF84 (for H-type BSE agent in BoPrP-Tg110 mice, PrP^res band measurements account for the overlapping of the two distinct glycosylated fragments). (A) PrP^res in hamsters. TgOvPrP4 ovine transgenic mouse-passaged SSBP/1 (1), C57BL/6-passaged classical BSE agent (2), L-type BSE agent (3), and TME agent passaged in cattle (4) are compared. (B) PrP^res in BoPrP-Tg110 mice. Classical BSE agent (1), H-type BSE agent (2), L-type BSE agent (3), and TME agent passaged in cattle (4) are compared. Error bars indicate standard deviations.

In summary, Syrian golden hamsters developed prion disease after intracerebral challenge with both the L-type BSE agent and the TME agent passaged in cattle, whereas the classical and H-type BSE agents failed to transmit disease at first passage from bovine brains. PrP^res detected in the brains of hamsters and bovinized transgenic mice inoculated with the L-type BSE agent or the TME agent passaged in cattle exhibited the same molecular characteristics (electrophoretic mobilities, 12B2 labeling, and glycoform ratios), consistent with our earlier observations of these two TSE sources transmitted in the TgOvPrP4 mouse line (1). In contrast, a clearly different PrP^res pattern was observed in hamsters infected with the C-type BSE agent from infected C57BL/6 mice, although similarly high levels of diglycosylated PrP^res were identified in the three bovine TSE sources in hamsters. Altogether, these consistent observations strongly reinforce the hypothesis of a cross-species, food-borne transmission of the L-type BSE agent as the origin of TME.

Our findings also show that the absence of transmission of the classical or H-type BSE agent from cattle to hamsters is the result of a “strain barrier” rather than a “species barrier” between cattle and hamsters, which could be readily bypassed by the L-type BSE agent or the TME agent passaged in cattle in our study. The lack of transmissibility of the classical BSE agent from cattle to hamsters and its efficient transmission after a first passage in C57BL/6 wild-type mice are consistent with the results of previous studies (11, 20, 21). Hamsters were also susceptible to the SSBP/1 isolate from ovine transgenic TgOvPrP4 mice, consistent with the known susceptibility of hamsters to some scrapie sources (14). Interestingly, opposite outcomes of transmissions were observed between cattle and wild-type mice, with efficient primary transmissions of the classical and H-type BSE agents (2) but not of the L-type BSE agent or the TME agent passaged in cattle (8; S. Nicot and T. G. Baron, unpublished data), whereas the four bovine TSE agents transmitted readily to bovinized transgenic mice. Overall, our results well corroborate the notion that the majority of the so-called species barriers are actually strain barriers (10, 18) and suggest that the prion seeds of the L-type BSE agent and the TME agent passaged in cattle might be more conformationally compatible with the structure of the hamster prion protein.

It was recently proposed that interspecies transmission of prions could be tightly controlled by the local β₂-α₂ loop region of the PrP protein encompassing amino acid residues 165 to 175 (19). In particular, the homology at position 170 (S or N) was shown to be critical for prion transmission. Cattle, sheep, and mice are 170S animals, whereas Syrian hamsters are 170N animals. The transmission obtained in hamsters in our study thus illustrates that specific prion strains can overcome the codon 170 homology requirement (19). Conversely, the L-type BSE agent is not transmissible to wild-type mice (8; Nicot and Baron, unpublished data), which are susceptible to both the classical and the H-type BSE agents (2), although mice and cattle share the 170S amino acid. It could be speculated that particular conformations of the β₂-α₂ loop region may be associated with the TSE agent involved in L-type BSE or/and that differences in other regions of the prion protein sequence might be critical for the interspecies transmission of this recently identified form of BSE in cattle.