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Heterogeneity of the Abnormal Prion Protein (PrP Sc) of the Chandler Scrapie Strain
research-article
Author(s):
Kazuo Kasai ,
Yoshifumi Iwamaru ,
Kentaro Masujin ,
Morikazu Imamura ,
Shirou Mohri ,
Takashi Yokoyama
*
Publication date (Electronic):
18 February 2013
Journal:
Pathogens
Publisher:
MDPI
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Abstract
The pathological prion protein, PrP Sc, displays various sizes of aggregates. In this study, we investigated the conformation, aggregation stability and proteinase K (PK)-sensitivity of small and large PrP Sc aggregates of mouse-adapted prion strains. We showed that small PrP Sc aggregates, previously thought to be PK-sensitive, are resistant to PK digestion. Furthermore, we showed that small PrP Sc aggregates of the Chandler scrapie strain have greater resistance to PK digestion and aggregation-denaturation than large PrP Sc aggregates of this strain. We conclude that this strain consists of heterogeneous PrP Sc.
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Most cited references24
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Identification of a protein that purifies with the scrapie prion.
N McKinley, D. Bolton, S Prusiner (1982)
Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.
- Record: found
- Abstract: found
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The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent
Philippe Tixador, Laetitia Herzog, Fabienne Reine … (2010)
Introduction Transmissible spongiform encephalopathies (TSE), such as human Creutzfeldt-Jakob disease, sheep scrapie, bovine spongiform encephalopathy (BSE) and chronic wasting disease of cervidae, are infectious, fatal, neurodegenerative disorders caused by prions [1]. Prions are unconventional pathogens primarily composed of PrPSc, a rearranged conformer of the ubiquitously expressed prion protein (PrPC), whose precise physiological function is largely unknown. Upon infection, PrPSc dictates the self-perpetuating conformational conversion of PrPC into nascent PrPSc. This conversion involves – without any apparent post-translational modification – the refolding of soluble, alpha-helix-rich PrPC molecules into beta-sheet enriched PrPSc polymers that form deposits in TSE-infected brains [2], [3] and are assumed to be responsible for the observed neurodegenerative disorders [4]. The conversion reaction may proceed through a nucleated polymerization mechanism in which PrPSc multimers recruit PrPC molecules and trigger their conformational conversion into PrPSc (for review [5]). The refolding/multimerisation process confers distinct physico-chemical properties to PrPSc, such as insolubility in non-denaturing detergents and partial resistance to proteolysis [6]. Distinct prion entities, referred to as strains, are known to self-propagate in the same host and exhibit distinguishable phenotypic traits that are heritable, such as incubation time, neuropathological and biochemical properties (for reviews: [7], [8], [9]). Accumulating experimental evidence indicates that strain-specified properties are encoded within structural differences in the conformation of the PrPSc molecules, which are faithfully imparted to host PrPC during the conversion process [10], [11], [12], [13], [14], [15], [16], [17]. However, the extent to which the aggregation state varies between different stains, and participates to strain-specific prion biology is unknown. The various fractionation methods and preparative procedures previously employed to estimate the size of the infectious particles [18], [19], [20], [21], [22], [23], [24], [25] have led to a vast range of measured sizes, making it difficult to relate any variation to potential strain differences. Of note, almost all of these studies used substantially purified PrPSc as a starting material. In this study, we developed a specific protocol to fractionate PrP particles according to their sedimentation velocity properties in a viscous medium, characterized their relative levels of infectivity and looked for strain-specific variations. In contrast to previous reports, experiments were performed on crude brain homogenates, which a priori contain all TSE infectivity. We worked with a panel of strains that were biologically cloned on homogeneous genetic backgrounds, obtained after transmission of either classical and atypical (Nor98) sheep scrapie and BSE, or hamster scrapie infectious sources in transgenic mice expressing ovine PrP (VRQ allele; tg338 mice) and hamster PrP (tg7 line), respectively. We demonstrate that the sedimentation profile of the infectious component dramatically varies with the strain. We further show that the predominance of slowly sedimenting infectious particles that segregate from the bulk of proteinase K-resistant PrPSc particles may be a distinctive feature of strains able to induce a rapidly lethal disease. Results Optimizing the conditions to analyze non-denatured PrPSc polymers by sedimentation velocity PrPSc aggregates present in detergent-solubilised brain tissue homogenates were fractionated by sedimentation velocity centrifugation in an iodixanol gradient (Optiprep). The experimental conditions were established with brain material from tg338 mice that were infected or not with LA21K fast strain (referred to as LA21K), a prototypal, rapid strain that kills the mice within ∼2 months (see Table 1 for information on the strains used in this study). As a first step, we tested a variety of detergents for solubilization, which showed variable efficacy in terms of partition of PrPC and PrPSc species. For example, the use of standard solubilization buffers containing Triton X-100 and sodium deoxycholate or sarkosyl led to sedimentation of both isoforms throughout the gradient ( Figure S1 ), indicating an incomplete release of total PrP from cellular constituents. In contrast, the sequential use of dodecyl maltoside and sarkosyl resulted in more efficient separation of the two PrP isoforms. Thus, in the conditions eventually employed (see Figure 1 for a summarizing flow diagram), the bulk of PrPC molecules remained in the upper fractions 1–4 ( Figure 2A and D , green line), while both PrPSc ( Figure 2B ) and proteinase K (PK) resistant PrPSc species ( Figure 2C–D , black line) were mainly detected in fractions 6–20 of the gradient. Importantly, no pelleted PrP material was observed in the selected conditions. Increasing the ultracentrifugation time caused the majority of PrPSc to sediment toward the heaviest fractions of the gradient, indicating that this material had not reached its density equilibrium (data not shown). Both dodecyl maltoside and sarkosyl are known to efficiently solubilize membrane structures, including rafts [26], [27], [28], yet PrPSc could be attached to abnormal, prion-induced structures. To address this point, brain homogenates were solubilized using these detergents in more stringent conditions, i.e. at 37°C instead of 4°C [29], however the sedimentation profile of PrPSc was affected only marginally ( Figure S2A ). 10.1371/journal.ppat.1000859.g001 Figure 1 Flow diagram describing the sedimentation velocity protocol and the analysis of prion particles infectivity with regard to PrPSc content. 10.1371/journal.ppat.1000859.g002 Figure 2 Immunoblot analysis of velocity sedimented PrP material from tg338 mouse brain. Brain homogenates from uninfected (A) or scrapie-infected (B–C; LA21K strain) tg338 mice were solubilized and fractionated by sedimentation velocity. The collected fractions (numbered from top to bottom of the gradient) were analyzed for PrP content by immunoblot without (A, B) or after PK digestion (C). (D) Graph showing the relative amount of PrPC (green line) and PK-resistant PrPSc (black line) per fraction. MM: molecular markers. 10.1371/journal.ppat.1000859.t001 Table 1 Phenotypic traits of the ovine and hamster prion strains used in the study. Strains1 Survival time2 PrPres pattern3 References Ovine LA21K (fast) 55±1 21 kDa Unpublished 127S 56±1 21 kDa [61] LA19K 133±3 19 kDa Unpublished BSE 135±3 20 kDa [60] Nor98 186±4 19 kDa+10 kDa [41] Hamster 139 H 36±1 21 kDa [67] and unpublished Sc237 45±1 21 kDa [67] and unpublished ME7H 141±3 21 kDa Unpublished 1 All but ME7H have been cloned by transmission at limiting dilution. 2 Measured in recipient transgenic mice expressing ovine PrP (tg338 line) or hamster PrP (tg7 line). Expressed as mean (in days) ± SEM. 3 As referred to the size of unglycosylated PK-resistant PrPSc in immunoblots. In order to assess the reproducibility of the partition and to enable quantitative analysis of the data, 7 independent fractionations were performed using different pooled or individual brains and the resulting data fitted ( Figure 3A , black line). This revealed that ∼80% of the PK-resistant PrPSc material sedimented as one major peak (maximum in fractions 10–12) with a Gaussian-like distribution. Standard globular macromolecules and ovine recombinant PrP oligomers [30] loaded on gradients run in parallel enabled estimation of the approximate molecular mass of the PK-resistant PrPSc aggregates forming the peak in fraction 10–12: between 200 and 500 kDa (by reference to the marker proteins, the sedimentation profile of which was affected only marginally in the presence of detergents), and ∼850 kDa based on the position of the 36-mer PrP oligomer ( Figure 3A ). 10.1371/journal.ppat.1000859.g003 Figure 3 Distinct PK-resistant PrPSc and infectivity sedimentation profiles of ovine prion strains. Brain homogenates from tg338 mice infected with LA21K (A), 127S (B), LA19K (C), Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analyzed for PK-resistant PrPSc content (black line) and for infectivity (red line). The mean levels of PK-resistant PrPSc per fraction have been obtained from the immunoblot or ELISA analysis of n = 4 to 7 (as indicated on each graph) independent fractionations. Since the replicates gave consistent results, these data were combined and fit. For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis) and by applying these values to standard dose response curves [34], established by inoculation of serial tenfold dilutions of mouse brain homogenates infected with the same strain (see Figure S5 and material and methods). In these titration experiments, animals inoculated with 2 mg of infectious brain tissue were assigned a relative infectious dose of 0. The right, blue logarithmic scale provides the strain-specific reciprocal relation between survival time and relative infectious dose. For all but 127S strain, the data presented are the mean of n = 2 independent titrations. The sedimentation peaks of standard molecular mass markers (MM markers) aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (669 kDa) and of 12-, 24-, and 36-mers of ovine recombinant PrP (recPrP) oligomers are indicated on the top of the graph. When solubilized brain material was PK-treated prior to ultracentrifugation, the PrPres sedimentation profile resembled that observed with intact brain material ( Figure S2B ). However, when semi-purified PrPSc in the form of scrapie-associated fibrils [31], [32] was resolubilized and centrifuged, a markedly different profile was obtained, with peaks in fractions 22 and 30 (bottom fraction) ( Figure S2C ). Interestingly, fast sedimenting PrPSc material was also observed with Italian scrapie agent (referred to as SSit), which in tg338 mice produces very long incubation times and abundant plaque-like PrPSc deposits in the brain [33], in contrast to the LA21K agent. These plaques can be stained by thioflavin S ( Figure S3A–B), indicating the presence of amyloid fibrils. When SSit-infected brain material was fractionated, the majority of PrPSc multimers peaked in fractions 24 to 30 of the gradient ( Figure S3C ). These results suggest that the experimental conditions employed preserve potential differences in the aggregation state of PrPSc thereby enabling the comparative analysis of sedimentation properties of “close to natural” PrPSc aggregates and of associated infectivity. Fast ovine prion strains have distinct infectivity and PK-resistant PrPSc sedimentation profiles The distribution of prion infectivity throughout the gradients was determined by an incubation time bioassay [34]. tg338 mice were inoculated intracerebrally with diluted aliquots from the different fractions. In terminally diseased mice, the PrPSc electrophoretic profile and regional distribution in the brain observed for representative fractions were both consistent and similar to that with the original brain material, indicating a conservation of the strain biological phenotype ( Figure S4A and data not shown). The mean survival time values resulting from the analysis of 2 independent gradients are shown in Figure 3A (red line). Typically, the mice inoculated with the PK-resistant PrPSc-richest fractions (6–20) succumbed to disease in more than 80 days, whereas those inoculated with fractions 1–3 died in a markedly shorter time, ∼60–70 days. The correlation between the mean survival time values and infectivity was established by using a standard infectious dose/survival time curve previously established for this strain ( Figure S5 ). This analysis indicated that fractions 1–2 were between 100- and 1000-fold more infectious than fractions 6–20 ( Figure 3A , blue scale). These upper fractions - within the sedimentation peak of aldolase (158 kDa) and upstream of 12-mer PrP oligomer – totaled 40 days). The most infectious sheep BSE fractions were found in fractions 6–12, 16 and 20 (mean survival times of 155–160, 164 and 163 days) while the top and bottom fractions were about 50-fold and 100-fold less infectious, respectively (survival time of ∼175 days and >180 days; Figure 3E ). Sedimentation properties of hamster prions To further explore the possibility that slow sedimenting infectivity could be a specific feature of fast prion strains, we applied the same sedimentation velocity protocol to three hamster strains passaged on tg7 transgenic mice expressing hamster PrP ( Table 1 ). For fast strains 139H and Sc237, the infectivity peaked in the top two fractions, which contained ∼10% of the total PK-resistant PrPSc material present in the gradient. The two 139H PK-resistant PrPSc peaks in fractions 11–12 and 16–18 and the Sc237 PK-resistant PrPSc peak in fraction 11–12 were ∼50-fold and 400 days post-infection. Their brain was negative for PrPres content. Mice showing TSE neurological signs were monitored daily and euthanized in extremis. Brains were removed and analyzed for PrPres content by either immunoblot or histoblot (see below) as a confirmatory test. The survival time was defined as the number of days from inoculation to euthanasia. The survival times of tg338 or tg7 reporter mice was measured for each tenfold dilution tested during endpoint titration experiments performed with all but ME7H strains. Animals inoculated with 2 mg of infectious brain tissue were assigned a relative infectious dose of 0. From these data, curves representing the relative infectious dose to survival time were established ([41] and Figure S5 ). The different patterns in survival time distribution among the gradients can thus be looked at as a function of relative infectious dose so as to estimate what difference in survival times between inoculated fractions means in terms of infectivity. Rov cell assay for infectivity titration The scrapie cell assay technique will be fully described elsewhere. Briefly, LA21K gradient fractions aliquots (typically 20–30 µL) were methanol precipitated before resuspension in culture medium (alpha minimal essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 10 µg/ml streptomycin). We verified that methanol precipitation did not affect the overall level of infectivity. Rov cell [40] monolayers established in a 96 well plate were exposed to the fractions for one week. After several washes, the cells were further cultivated for two weeks before fixation and PrPSc detection by immunofluorescence as previously described [65]. Immunofluorescence signals were acquired with an inverted fluorescence microscope (Zeiss Axiovert). A program in NIH Image J software was designed to quantify the levels of PrPSc signal per cell in each well. Serial tenfold dilutions of LA21K infected brain homogenates were prepared in the same conditions and run in parallel experiments to establish a tissue culture infectious doses curve that directly relates to the percentage of PrPSc content. Histopathology Brains were rapidly removed from euthanized mice and frozen on dry ice. Cryosections were cut at 8–10 µm, transferred onto Superfrost slides and kept at −20°C until use. Histoblot analyses were performed on 3 brains per infection, using the 12F10 anti-PrP antibody as described [60]. For thioflavin-S binding, formalin- or methanol-fixed sections were incubated with 0.01% thioflavin-S for 1 hour as previously described [66]. Sections were then incubated with nuclear marker 4′, 6-diamidino-2-phenylindole (Sigma), mounted in fluoromount-G (Interchim) before acquisition with an inverted fluorescence microscope (Zeiss Axiovert) and analysis with the Metamorph software. Accession numbers The Swiss-Prot accession numbers for the proteins mentioned in the text are sheep (P23907) and hamster PrP (P04273). Supporting Information Figure S1 Effects of the detergents used to solubilize brain homogenates on the sedimentation properties of PrPC and PrPSc molecules. Uninfected (A, C) or LA21K infected (B, D) brain homogenates (20% wt/vol.) were solubilized by adding an equal volume of standard lysis buffer (1% sodium deoxycholate, 1% Triton X-100, 100 mM Tris-HCl pH 7.4; A–B) or by 2% sarkosyl (C–D) for 30 min at 4°C. A volume of 150µl was loaded atop a iodixanol gradient (5–25% Optiprep in 25mM HEPES, 150mM NaCl, 1∶2 dilution of standard lysis buffer (A–B) or 1% sarkosyl (C–D)) and centrifuged at 200 000 g for 60 min at 4°C in a SW55 rotor. Fifteen fractions were collected and analyzed for PrPC (A, C) and PK-resistant PrPSc (B, D) content by immunoblot. Fractions were numbered from top to bottom of the gradient. (2.01 MB TIF) Click here for additional data file. Figure S2 PrPSc sedimentation velocity profile following solubilization at 37°C, PK digestion or aggregation. (A) LA21K brain homogenate was solubilized in the same conditions as in the standard protocol (see Figure 1), except that the temperature was increased to 37°C. The resulting solution was sedimented by velocity. (B, C) LA21K brain homogenate was either digested with 100 µg/ml of PK for 1h at 37°C (B) or subjected to a “scrapie-associated fibrils” protocol (C, see Methods) before applying the standard fractionation protocol (see Figure 1). All the collected fractions were analyzed for PK-resistant PrPSc content by immunoblot. For each fraction, the percentage of the total sum of all PK-resistant PrPSc detected on the immunoblot is presented. (0.43 MB TIF) Click here for additional data file. Figure S3 Sedimentation velocity of Italian scrapie agent. (A, B) Nuclear marker 4′, 6-diamidino-2-phenylindole (DAPI, red) and thioflavin S staining (green) of coronal brain sections from mice infected with Italian scrapie (SSit; A) or LA21K (B) agent. Note that in SSit-infected brains, thioflavin S positive plaques were distributed in a rosary-like array along notably the corpus callosum. (C) Graph showing the relative amount of SSit PK-resistant PrPSc per fraction after fractionation of infected brain homogenate in the standardized conditions (see Methods). (3.37 MB TIF) Click here for additional data file. Figure S4 Regional distribution of PrPSc deposits in the brains of tg338 mice inoculated with sedimentation velocity fractionated brain homogenates. Tg338 mice were infected intracerebrally with either crude or fractionated LA21K-infected brain homogenate (A) or fractionated, sheep BSE-infected brain homogenate (B). The PrPSc deposition pattern in the brains of inoculated mice was examined by histoblot analysis as previously described [60]. (A) The intensity of PrPSc deposition in several brain regions was scored. (B) The distribution of PrPSc deposits in mice brains is shown on representative histoblots of 4 different antero-posterior sections. Note that the staining observed after inoculation of top, middle and bottom fractions were similar and reminiscent of that previously reported after inoculation of different BSE-related agents in tg338 mice [60]. (5.23 MB TIF) Click here for additional data file. Figure S5 Titration of ovine and hamster prion strains infectivity. (A) Survival time of tg338 mice intracerebrally inoculated with serial tenfold dilutions of brain homogenate from LA21K-infected tg338 mice. The mean values measured, the SEM (error bars) and the number of diseased/inoculated mice for each dilution are indicated on the right of the plot. Animals inoculated with the equivalent of 2 mg of infectious brain tissue were assigned a relative infectious dose of 0. The diseased mice were positive for brain PrPres. A regression curve has been drawn from the mean survival times measured. (B) From this curve, levels of infectivity expressed (y, in Log (infectious dose)) can be determined from survival times values (x, in days), using the equation fit to the data. The constants of the equation are also indicated for all strains for which an endpoint titration was available. (0.17 MB TIF) Click here for additional data file. Figure S6 Thermolysin-resistance and insolubility of the PrP species present in LA21K upper fractions. Uninfected (−) or LA21K-infected (+) brain homogenates (A) or pooled fractions 1–2 (B) were treated with thermolysin for 1 h at 70°C at the indicated concentrations, before immunoblotting with either Sha31 or Pc248 anti-PrP antibodies, the latter being directed against the N-terminal part of PrP. (C) The top six fractions from an uninfected or LA21K-infected gradient were treated with thermolysin (125 µg/ml final concentration) for 1 h at 70°C before analysis by immunoblotting with Pc248 antibody. After measurement of chemoluminescence intensities and normalization as referred to total protein content, the ratio of LA21K infected to uninfected signal was calculated for each fraction to determine the presence of thermolysin-resistant PrPSc. The results represent the mean ± SEM of 4 independent fractionations, analyzed in duplicate. (D) Fractions from uninfected and LA21K-infected gradients were ultracentrifuged at 100 000 g for 1 h at 4°C to generate soluble (supernatant) and insoluble (pellet) fractions, before immunoblot analysis. After measurement of chemoluminescence intensities, the ratio of LA21K infected to uninfected signal was calculated for the pellet and supernatant of each fraction (after normalization of total protein content). The results represent the mean ± SEM of 4 independent fractionations. (E) A pool of 1–2 fractions from an uninfected (−) or LA21K-infected (+) gradient were ultracentrifuged at 100 000 g for 1 h at 4°C to generate supernatant (S) and pellet (P) fractions, before immunoblot analysis. Note that the vast majority of post-fractionated PrPC remained associated with the soluble fraction, suggesting efficient solubilization. (1.46 MB TIF) Click here for additional data file. Figure S7 Quantification of LA21K infectivity sedimentation profile by Rov cell assay. The distribution and level of LA21K infectivity in the gradient was measured using a Rov cell [40] assay (JC, VB, HL, unpublished data). This assay is based on the detection of PrPSc-containing Rov cells by immunofluorescence using PrPSc-specific antibodies. Rov cells were exposed in parallel to fraction aliquots and to serial tenfold dilutions (expressed as relative infectious doses as in Figure 3) of a LA21K-infected brain homogenate prepared in the same conditions. The culture and PrPSc detection conditions have been optimized to enable a quantitative relationship between the percentage of PrPSc content (± SEM) and LA21K infectious titer. The data presented are the mean of n = 2 independent titrations. (0.24 MB TIF) Click here for additional data file.
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Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.
Elizabeth R. Marsh, Richard A. Bessen (1992)
Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.
Author and article information
Journal
Journal ID (nlm-ta): Pathogens
Journal ID (iso-abbrev): Pathogens
Journal ID (publisher-id): pathogens
Title:
Pathogens
Publisher:
MDPI
ISSN
(Electronic):
2076-0817
Publication date
(Electronic):
18
February
2013
Publication date Collection:
March
2013
Volume: 2
Issue: 1
Pages: 92-104
Affiliations
Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki
305-0856, Japan; E-Mails:
kakasai@
123456affrc.go.jp
(K.K.);
gan@
123456affrc.go.jp
(Y.I.);
masujin@
123456affrc.go.jp
(K.M.);
imamuram@
123456affrc.go.jp
(M.I.);
shirou@
123456affrc.go.jp
(S.M.)
Author notes
[*
]Author to whom correspondence should be addressed; E-Mail:
tyoko@
123456affrc.go.jp
(T.Y.); Tel.: +81-29-838-7840; Fax: +81-29-838-8332.
Article
Publisher ID: pathogens-02-00092DOI: 10.3390/pathogens2010092
PMC ID: 4235706
SO-VID: 7c44f231-38a3-479f-8955-0c6ac936f5be
Copyright © © 2013 by the authors; licensee MDPI, Basel, Switzerland.
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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).
History
Date
received
: 09
January
2013
Date
revision received
: 07
February
2013
Date
accepted
: 09
February
2013
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