Volume 23, Number 6—June 2017
Distribution and Quantitative Estimates of Variant Creutzfeldt-Jakob Disease Prions in Tissues of Clinical and Asymptomatic Patients
|Tissue||Clinical vCJD patients, Met129/Met129
||Preclinical vCJD patient, Met129/Val129
||Non–vCJD controls, Met129/Met129
|Dorsal root ganglia||NA||NA||NA||NA||–||–||–|
|Cervical lymph node||10−5||10−4||10−4||10−3||10−4||NA||NA|
*PMCA reactions were seeded with 10-fold serial dilutions of 10% tissues homogenates (10−2–10−9) that had been collected postmortem from 4 symptomatic vCJD patients (vCJD-1–vCJD-4) or an asymptomatic vCJD-infected person (vCJD-A). At least 4 replicates of each sample dilution were tested in 2 independent PMCA experiments. Prions from patients vCJD-1–vCJD-4 were homozygous for methionine at codon 129 of the PRNP gene. Prion from patient vCJD-A was heterozygous (methionine/valine) at codon 129 of the PRNP gene. PMCA substrate was prepared by using brains from transgenic mice overexpressing the ARQ variant of sheep prion protein. Reactions seeded with tissues from 2 non–vCJD-infected control patients (NC-1 and NC-2) were included as negative controls. PMCAs were subjected to 4 rounds of amplification, each composed of 96 cycles (sonication for 10 s and incubation for 14.5 min at 39.5°C) in a Qsonica700 Sonicator (Qsonica LLC, Newtown, CT, USA). PMCA reactions were analyzed by Western blotting for proteinase K–resistant PrP by using Sha31 antibody epitope YEDRYYRE. Values are the highest dilution that resulted in a positive Western blot result in >50% of the tested replicates after 4 PMCA amplification rounds. NA, not applicable; PMCA, protein misfolding cyclic amplification; PrP, prion protein; vCJD, variant Creutzfeldt-Jakob disease; –, negative.