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PRNPThe methionine/valine (M/V) SNP at codon 129 of the prion protein gene PRNP influences susceptibility to prion diseases. Diack et al. report that vCJD strain properties are not affected by transmission through an individual with the PRNP MV codon 129 genotype, suggesting no association between this genotype and altered virulence.
Abstract
In 2004, a subclinical case of variant Creutzfeldt-Jakob disease in a PRNP 129 methionine/valine heterozygous individual infected via blood transfusion was reported, and we established that the spleen from this individual was infectious. Since host genetics is an important factor in strain modification, the identification of variant Creutzfeldt-Jakob disease infection in a PRNP 129 methionine/valine heterozygous individual has raised the possibility that the properties of the variant Creutzfeldt-Jakob disease agent could change after transmission to this different genetic background and concerns that this could lead to a more virulent strain of variant Creutzfeldt-Jakob disease. The variant Creutzfeldt-Jakob disease strain has to date been characterized only in methionine homozygous individuals, therefore to establish whether the strain characteristics of variant Creutzfeldt-Jakob disease had been modified by the host genotype, spleen material with prion protein deposition from a PRNP 129 methionine/valine individual was inoculated into a panel of wild-type mice. Three passages in mice were undertaken to allow stabilization of the strain characteristics following its passage into mice. In each passage, a combination of clinical signs, neuropathology (transmissible spongiform encephalopathy vacuolation and prion protein deposition) were analysed and biochemical analysis carried out. While some differences were observed at primary and first subpassage, following the second subpassage, strain characteristics in the methionine/valine individual were totally consistent with those of variant Creutzfeldt-Jakob disease transmitted to 129 methionine/methionine individuals thus demonstrated no alteration in strain properties were imposed by passage through the different host genotype. Thus we have demonstrated variant Creutzfeldt-Jakob disease strain properties are not affected by transmission through an individual with the PRNP methionine/valine codon 129 genotype and thus no alteration in virulence should be associated with the different host genotype.
Introduction
Variant Creutzfeldt-Jakob disease (vCJD) is an acquired prion disease linked to the consumption of food products contaminated with the bovine spongiform encephalopathy (BSE) agent (Bruce et al., 1997; Hill et al., 1997) and was first reported in the UK in 1996. A peak in deaths was recorded in 2000 and it has since declined with a total of 178 deaths between 1995 and 2018 in the UK (Will et al., 1996; National CJD Research and Surveillance Unit, 2019).
Until 2016, all definite and probable cases of vCJD with genotype data had occurred in the 129 methionine homozygous (MM) genotype suggesting other genotypes may be more resistant to vCJD. In 2016 the first case of clinical vCJD in a 129 methionine/valine heterozygous (MV) individual was reported demonstrating that individuals with other genotypes were susceptible to vCJD and raising the possibility of a second wave of vCJD in individuals of this genotype (Mok et al., 2017). Transmission studies in transgenic mice expressing human prion protein (PrP, encoded by PRNP) had predicted that all three genotypes were susceptible to vCJD albeit with differences in susceptibility and incubation period. The 129MV and 129VV genotypes were predicted to have longer incubation periods than in the 129MM individuals and indeed 129MV and 129 valine homozygous (VV) individuals may not develop clinical signs of disease (Bishop et al., 2006). However, since vCJD infection can reside in peripheral tissues there is potential for onward transmission of infection from individuals of all three genotypes (Bruce et al., 2001; Ritchie et al., 2009).
Human to human transmission of vCJD has already been demonstrated and while the majority of vCJD cases are primary cases presumed to be acquired from BSE, three clinical cases of vCJD have been identified in 129MM individuals who had received non-leucoreduced red blood cell concentrates from asymptomatic UK donors (Llewelyn et al., 2004; Hewitt et al., 2006; Wroe et al., 2006). Evidence of misfolded PrP in peripheral tissues has been identified in two other individuals linked to blood and blood products, both of whom were of the 129MV genotype and remained asymptomatic until death from non-vCJD related causes (Peden et al., 2004, 2010). Studies on one of these individuals who had received a red blood cell transfusion from a 129MM donor showed no evidence of abnormal PrP in the brain but deposition in the spleen and a cervical lymph node was observed (Peden et al., 2004) and using protein misfolding cyclic amplification (PMCA), prion seeding potential was demonstrated in a range of tissues (Bougard et al., 2016). Bioassay of the spleen material from this individual also confirmed the presence of vCJD infectivity (Bishop et al., 2013). The other 129MV individual was an adult haemophilic patient who had received factor VIII concentrate prepared from plasma pools known to include a donation from a vCJD infected donor. In this case, spleen material tested positive for the presence of PrP by western blot analysis (Peden et al., 2010).
Three retrospective studies of anonymized human appendix samples have been carried out in the UK to ascertain the prevalence of vCJD infection (Hilton et al., 2004; Ironside et al., 2006; Gill et al., 2013; Public Health England, 2016). The most recent study identified seven positives out of 15 939 giving a prevalence of ∼1:2000 individuals carrying abnormal PrP (Public Health England, 2016). In these three retrospective studies, misfolded PrP has been found in all three codon 129 genotypes.
The single nucleotide polymorphism at codon 129 of the prion protein gene, PRNP, encoding either M or V, is recognized as influencing host susceptibility and modifying strain characteristics for human prion diseases such as Creutzfeldt-Jakob disease, Kuru, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia (Lee et al., 2001; Pocchiari et al., 2004; Kobayashi et al., 2015). It was thus recognized that vCJD in different host genotypes may display different strain characteristics.
We have reported previously the infectivity of spleen tissue from an asymptomatic MV blood recipient (MV) and the spleen and brain tissue of the MM donor to that individual (MM) in RIII and transgenic mice (Bishop et al., 2013). The transmission of MV and spleen and brain homogenate of the MM into RIII mice also demonstrated differences in incubation times and attack rates between the two inocula suggesting either differences in titre of infectivity or different strain properties between MM and MV (Bishop et al., 2013). It was therefore important to determine the underlying reason for these differences observed on the initial passage to mice, since strain differences could impact on the transmission potential of the strains.
The MV case demonstrates that there is the risk of transmission between asymptomatic individuals of the MV genotype through blood transfusion or blood products. With the possibility of strain modification and adaptation of the vCJD agent following transmission to a different genotype, it is thus important to assess whether vCJD is modified by the genetic background and whether there are alterations in virulence or pathogenesis. To address this we have carried out an extensive stain typing analysis on the strain of agent found within the spleen of an asymptomatic codon 129MV individual. We have established that the characteristics of this strain are consistent with that of vCJD from 129MM individuals. Thus we have demonstrated that strain properties of vCJD are not altered by the PRNP codon 129 genotype of an individual.
Materials and methods
Human tissue selection
Post-mortem tissue (brain and spleen ∼0.5-1.5 g) was collected via the MRC Edinburgh Brain Bank, approved by a national ethics committee, in line with the Human Tissue (Scotland) Act. Use of human tissue for post-mortem studies has been reviewed and approved by the Sudden Death Brain Bank ethics committee and the Academic and Clinical Central Office for Research and Development (ACCORD) Medical Research Ethics Committee (AMREC). Clinical and demographic details are given in Table 1. Tissue samples were homogenized at 10% (w/v) concentration in sterile physiological saline and stored at −80°C until use.
Demographic and clinical features of cases included in this study
Mice and experimental design
A panel of three inbred wild-type mouse lines were used for the transmission studies; RIII (also referred to as MR), C57BL6 and VM. RIII and C57BL6 lines are of the Prnp genotype and VM are of the Prnp genotype (Bruce et al., 1991). This combination of mouse lines has been used for the strain characterization of a number of human and animal prion diseases and each line has a characteristic and reproducible incubation period and pathology when inoculated with vCJD.
To characterize the isolates of interest fully, a primary pass (human to mouse) followed by two subpassages (mouse to mouse) were carried out. Subpassage was undertaken from a representative mouse from each mouse line that showed clinical signs and had positive transmissible spongiform encephalopathy (TSE) pathology (TSE vacuolation or PrP deposition) where possible. Brain material from this mouse was then inoculated into the panel of wild-type mice; this process was repeated for the second subpassage.
vCJD infection of mice
All animal studies were carried out in derogated CL3 facilities. The mice were housed in individually ventilated cages under a 12-h light/dark cycle and given food and water ad libitum. Cohorts of mice (n = 18-24, 6-8 weeks of age and sex matched) were given prophylactic antibiotics (500 µl streptomycin and 500 IU penicillin) prior to inoculation with human isolates. Mice were anaesthetized with isoflurane and inoculated with 0.02 ml of 10% homogenate via the intracerebral (i.c) route and 0.1 ml via the intraperitoneal route. The MM recipient (MM) CNS was inoculated by the intracerebral route only because of limited tissue availability. For subpassage, inocula were prepared with mouse brain tissue at 1% (w/v) in physiological saline and mice inoculated with 20 µl via the intracerebral route only. Tissue samples were irradiated prior to second subpassage because of an animal house move. Animal studies were approved by The Roslin Institute’s Animal Welfare Ethical Review Board and were conducted according to the regulations of the 1986 United Kingdom Home Office Animals (Scientific Procedures) Act.
Scoring of clinical TSE disease and pathology
Mice were scored weekly for clinical signs from 100 (primary pass) or 50 (subpassage) days post inoculation (dpi) by operators blind to isolate/cohort combination according to a previously established TSE clinical scoring system (Dickinson et al., 1968). Mice were scored as unaffected, possibly affected or definitely affected using standard criteria. Any unusual clinical signs were noted and in older animals signs of ageing (loss of body condition, reduced activity) were taken into account. Mice were sacrificed after (i) two consecutive scores of definitely affected; (ii) after receiving scores of definitely affected in 2 of 3 weeks, or (iii) significant deterioration of condition. Mice with no signs of clinical disease were maintained until ∼700 dpi or until study termination.
Mice were sacrificed by cervical dislocation, the brains removed and cut sagitally with one half frozen in liquid nitrogen for biochemistry and the remaining half fixed in formal saline for 48 h and decontaminated with formic acid when required, prior to paraffin embedding. Coronal sections were cut (6 µm) and stained with haematoxylin and eosin and TSE-specific vacuolation was quantitatively scored blind to isolate and mouse line by a standard method in nine grey matter and three white matter areas (Fraser and Dickinson, 1968). Mean TSE vacuolation scores were plotted for groups of six mice or more unless otherwise stated.
Prion protein detection by immunohistochemistry
Coronal sections were stained using 6H4 antibody (1:500, Prionics) (Korth et al., 1997) to detect PrP. Antigen retrieval by autoclaving at 121°C in citric acid buffer and a 5-min formic acid (98%) treatment was used. Sections were then blocked with normal rabbit serum prior to incubation with the primary antibody. Antibody binding was detected with the Vector ABC kit (Vector Laboratories) and visualized with 3,3′ diaminobenzidine chromogen. All sections were counterstained with haematoxylin.
Western blot analysis
Frozen brain samples were homogenized in NP40 buffer [20 mM Tris (pH 7.4), 0.5% v/v NP40 solution, 0.5% w/v sodium deoxycholate] to give a 10% w/v suspension. The suspension was further passaged through a 25 G needle (twice) to break up any remaining aggregates. The homogenate was combined with 3% SDS and treated with 20 μg/ml proteinase K (Novagen) for 1 h at 37°C with shaking.
The prepared sample was suspended in equal volume of Laemmli 2× sample buffer (Sigma-Aldrich) and treated at 100° for 10 min. The products were then loaded onto a 4-12% Bis/Tris gel (Thermo Fisher). After electrophoresis the gel was blotted onto a PVDF membrane. Detection of PrP was with primary antibody 6H4 (Prionics) (Korth et al., 1997) at 1:5000 (for a minimum of 24 h at 4°C with shaking) and an anti-mouse infrared fluorescence, IRDye® secondary antibody (LI-COR) at 1:5000. Images were captured with the LI-COR Odyssey imaging system.
Data and statistical analysis
Animals in which clinical signs were present without pathological (TSE vacuolation and/or PrP deposition) confirmation were removed from the analysis as these signs can also be due to other conditions such as ageing. Animals in which no TSE vacuolation score was available (due to tissue autolysis or technical issues) were also discounted. Early intercurrent deaths (under 200 dpi and 50 dpi for the primary and subpassage, respectively) were excluded from the study. Raw data and samples from previously published studies (Bishop et al., 2008; Bishop et al., 2013) were reanalysed using the criteria above to ensure a valid comparison between all datasets.
Statistical analyses for incubation periods were performed using A Kruskal-Wallis test followed by Dunn’s multiple comparisons test using GraphPad Prism 7.0 for windows (GraphPad Software, La Jolla California, USA).
Data availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
Results
In this study, we carried out a characterization study of the MV and MM (CNS and spleen) at primary passage in our strain typing panel of wild-type mice. This panel consists of RIII and C57BL6 mice, both of the Prnp genotype and VM mice, Prnp genotype. This combination of mouse lines gives highly reproducible and characteristic incubation periods (time between inoculation and death with clinical signs), incubation period rankings (order in which mouse lines succumb to disease) and neuropathology (TSE vacuolation and/or PrP deposition) when inoculated with the BSE or vCJD prion strain. These results were compared to a previously published transmission of brain homogenate from a MM blood donor (MM) and the associated MM blood recipient (MM) (Bishop et al., 2008). We then carried out a second passage of MV, MM (CNS and spleen), MM and MM and finally a third passage of the MV, MM (CNS) and MM to allow stabilization and full characterization of each strain.
Primary passage identified differences between isolates
The inoculation of MV (spleen) and MM (spleen and CNS) to the C57BL6 and VM mice resulted in positive transmission with both clinical disease and/or TSE vacuolation present in all isolate/mouse line combinations (Table 2). These data were then analysed and compared with the previously reported MVand MM RIII transmission and the MM and MMdataset.
Incidence of clinical disease and TSE vacuolation wild-type mice challenged with vCJD cases
On primary passage there were differences in attack rates for clinical signs and TSE vacuolation between the isolates with lower rates observed in both the MV and MM (spleen) across all mouse lines when compared to the MM (CNS). In particular, there was only one VM mouse scoring as clinically positive with no TSE vacuolation (PrP deposition was present) from the MV, compared to 47% with TSE vacuolation from the MM (spleen) and 54% with TSE vacuolation in previously-reported spleen transmissions from MM individuals (Ritchie et al., 2009).
Incubation periods were calculated for each line and isolate combination for mice exhibiting both clinical signs and TSE vacuolation and the incubation period ranking compared between isolates and those of previous transmissions (Fig. 1A). Incubation periods were increased for both MV and MM (spleen) in the RIII, C57BL6 and VM mice when compared to those of the MM (CNS). The incubation period ranking for the MM (CNS) was of the order RIII
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