Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.

Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.     

Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent

The scrapie agent causes a progressive degeneration of the central nervous system of animals after a prolonged incubation period. Measurements of incubation period length, defined as the time from inoculation to the onset of clinical signs of neurological dysfunction, were related to the titer of the agent and the dilution of the inoculated sample. Equations defining the relationship provide a new assay for the agent requiring fewer animals than end point titrations. By use of this incubation period assay, the scrapie agent from hamster brain was found to have an s20,w of < 300 S but > 30 S assuming rho p = 1.2 g/cm3. A partially purified fraction P3 was obtained by differential centrifugation and sodium deoxycholate extraction. When P3 was extracted with phenol, virtually no infectivity was found in the aqueous phase even after examining such variables as pH, salt concentration, and predigestion of samples with proteinase K. Nonionic and nondenaturing, anionic detergents did not inactivate the scrapie agent; in contrast, denaturing detergents inactivated the agent. Sodium dodecyl sulfate (NaDodSO4) inactivated greater than 90% of the agent at a NaDodSO4 to protein ratio of 1.8 g/g. Inactivation by NaDodSO4 appears to be a cooperative process. Addition of a nonionic detergent to form mixed micelles with NaDodSO4 prevented inactivation of the agent by NaDodSO4. Weak chaotropic ions do not inactivate the scrapie agent while strong chaotropic ions like SCN- and Cl3CCOO- destroy infectivity at concentrations of 0.2 M. These data provide evidence in support of a protein component within the scrapie agent which is essential for maintenance of infectivity. Thus, it is unlikely that the scrapie agent is composed only of a "naked" nucleic acid as is the case for the plant viroids.     

Linkage of prion protein and scrapie incubation time genes

A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW/LacJ mice have a short one (113 +/- 2.8 days). (NZW X I/Ln)F1 hybrid mice had incubation times of 223 +/- 2.8 days indicating longer incubation times were dominant. Incubation periods in the backcross progeny of (NZW/LacJ X I/LnJ)F1 X NZW/LacJ segregated into two groups (64 mice, 130 +/- 1.1 d; 66 mice, 195 +/- 1.9 d) indicating single gene control. NZW/LacJ and 20 other inbred strains have the Prn-pa allele which is identified as a 3.8 kb Xbal fragment using a hamster PrP (prion protein) cDNA probe. I/LnJ and three other Prn-pb mouse strains have a 5.5 kb Xbal restriction fragment. Analysis of DNA from 66 backcross mice indicated Prn-i is tightly linked to Prn-p, the structural gene for PrP.     

Immunological comparison of scrapie-associated fibrils isolated from animals infected with four different scrapie strains.

Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.     

Mouse-adapted ovine scrapie prion strains are characterized by different conformers of PrPSc

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.     

Immunological analysis of host and agent effects on Creutzfeldt-Jakob disease and scrapie prion proteins.

Creutzfeldt-Jakob disease (CJD) and scrapie are degenerative neurological diseases caused by unusual infectious pathogens. The term prion has been introduced to underscore the apparent distinctness of these agents from viruses and viroids. The only macromolecule shown to be associated with the infectious agent, the CJD or scrapie prion protein (PrPCJD or PrPSc, respectively), is encoded by the same gene as a normal cellular protein. In several studies biochemical differences have been reported in PrPScs derived from a common host species infected with different putative strains of the scrapie agent, suggesting agent-specific characteristics independent of the host. We analyzed various agent-host combinations by Western blotting of PrPs that were separated by size or charge. The profile of immunoreactive proteins for CJD prions isolated from mice, guinea pigs, and humans appeared distinct. Importantly, PrPCJDS purified from a human brain and from the corresponding first-passage mouse brains were clearly distinguishable. PrPCJDs isolated from CJD prions propagated in NAMRU or B10.Q mice, which are homozygous for a short-incubation-time gene; from the short-incubation-time backcross progeny of (B10.Q x I/LnJ)F1 x B10.Q; or from NAMRU mice inoculated with I/LnJ prions were identical to each other but distinguishable from those of I/LnJ mice, which are homozygous for the long-incubation-time gene. The PrPs from human CJD and ovine scrapie propagated in the same mouse strain appeared the same, but they were distinct from the same isolate of scrapie passaged in hamsters. Lastly, PrPScs purified from five different strains of scrapie propagated in C57BL mice were identical, including strains, ME7 and 139A, which were previously reported to be distinct. This evidence does not support, although it does not exclude, agent-mediated characteristics independent of host-mediated ones for scrapie and CJD.     

Disease phenotype in sheep after infection with cloned murine scrapie strains

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2–4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.     

Characterization of scrapie agent isolated from sheep in Japan

A pathogenic agent isolated in mice from the brain of a sheep affected by scrapie-like disease was characterized. The incubation period of the disease in the primary transmission from the sheep to mice was longer than in the secondary and the tertiary transmission in the same strain of mice. Progressive dilution of the inoculum caused prolongation of the incubation period. The infectivity of the agent in a 10% brain homogenate persisted, but decreased about 10(3) to 10(4) times after heating at 100 C for 30 min. Histological changes in the diseased mouse brains consisted of vacuolation of the nerve cells and spongiform degeneration in the gray matter of the central nervous system. Fine rod-shaped granulae with a length of 3 to 5 nm were observed within the swollen neuropil, axon, and perivascular astrocytic process. No serum antibodies against available mouse viruses, parainfluenza type 1 virus, lymphocytic choriomeningitis virus, and mouse reovirus type 3, were detected in any mice used in the experiments. These findings demonstrate that the disease of the sheep was the first case of scrapie in Japan.     

Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.     

Immune system-dependent and -independent replication of the scrapie agent.

Using the severe combined immunodeficiency (SCID) mouse model, we investigated the requirement of the immune system for the development of scrapie after peripheral inoculation. A total of 33% of SCID mice, all but one immunologically reconstituted SCID mice (93%), and all CB17 control mice developed the disease. PrPres was detectable in the brains of all diseased animals and in the spleens of reconstituted SCID and CB17 control mice but not of the diseased non-immunologically reconstituted SCID mice. The immune system appears to be a primary target in the pathogenesis of scrapie, but direct spread to the central nervous system from the peritoneum via visceral nerve fibers can probably also occur.     

Karyological characteristics of continuous mouse cell lines infected with the scrapie agent

A karyological study of murine cell line L, latently infected with different strains of the scrapie agent (Compton and C-506) showed that the both variants number of chromosomes and the number of Robertson's translocations of chromosomes decrease insignificantly with an increase in the time of subcultivation. The use of the C-method of differential chromosome staining revealed four new analogous chromosomes in both experimental cell lines. This phenomenon is probably specific for this infected cell model.     

Isolation and structural studies of the intact scrapie agent protein

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.     

Intraspecies prion transmission results in selection of sheep scrapie strains

Background

Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined.

Methodology/Principal Findings

In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain.

Conclusions/Significance

Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.     

An aphid-ant interaction: effects on different trophic levels

The interaction between the aphid Aphis coreopsidis (Thomas) (Hemiptera, Aphididae) and the ant Camponotus sp.1 (Hymenoptera, Formicidae) on the plant Bidens pilosa L. (Asteraceae) was studied. We manipulated the presence of ants and other insects to understand the effects of this aphid-ant interaction on: (a) the growth of A. coreopsidis population, (b) the seed production of B. pilosa, (c) the density of predator spiders. The growth of the A. coreopsidis population was positively affected by attending ants, showing greater increase on ant-present control plants than on ant-excluded plants. The density of aphids on control plants was positively correlated with the density of attending ants, while the density of aphids on ant-excluded plants was positively correlated with the density of spiders. Plants with ants had a significantly lower quantity of viable seeds than those without ants. These results show that: (a) the presence of Camponotus ants reduces the number of predator spiders on B. pilosa, (b) the population of A. coreopsidis increases when attended by the ants, (c) this increase in aphid density as a result of ant attendance reduces the number of viable seeds of B. pilosa, and thus (d) an interspecific interaction between two species can have an indirect negative fitness effect on other partners.     

The leg strength of two commercial strains of meat chicken subjected to different incubation profiles

Lower egg shell temperatures (EST) during the first 2 weeks of incubation, notionally known as Slow start incubation, extended the standing time of a 5-week-old fast feathering meat chicken parent line. This study was designed to evaluate the effect of Slow start incubation on the standing ability of commercial meat chickens. Eggs from two strains of meat chickens, Strains 1 and 2, were incubated using either the Slow start incubation, (the initial EST was 36.75°C followed by a gradual increase in EST, reaching 37.8°C at day 16 of incubation), or Control incubation (EST 37.75°C to 38°C from the start of incubation until day 18 of incubation). Eggs were observed every 6 h from 468 h until 516 h of incubation to identify chick hatch window. At 516 h of incubation all chicks were taken out of the incubator (take-off). Chicks from each Strain and incubation treatment were randomly selected for assessment of chick weight, chick length, yolk sac weight, serum Ca and P, and femoral bone ash (BA). All unhatched eggs were inspected to determine the stage of embryo failure. Remaining chicks were grown for 5 weeks in floorpens. Weekly feed intake (FI), chick weight and feed conversion ratio (FCR) were determined. At 35 days of age the standing ability of visibly male birds was assessed in a latency-to-lie test. Compared to the Control, Slow start incubation delayed the average hatch time of both strains by ∼13 h, and reduced hatchability with 4.6% live but unhatched chicks, which was most evident in Strain 2. Significant differences due to main effects only were observed at take-off. Strain 1 chicks were significantly heavier and longer with higher serum Ca but significantly lower BA and serum P than Strain 2. Slow start incubation generated significantly heavier chicks that were shorter, but had significantly heavier yolk sacs, lower serum Ca but higher serum P than Control incubated chicks. During the 1^st week post hatch Strain 1 Control incubated chicks had significantly higher FI and higher FCR than all other Strain and incubation treatments. At 35 days of age Slow start incubated birds of both Strains stood significantly longer than those from the Control incubation. This experiment clearly demonstrated the ability of Slow start incubation of commercial meat chickens to improve their leg strength.