Application Of RT-QuIC To Identification Of VPSPr And CWD Detection Of Skin Prion Protein

Prion disease is an infectious and fatal degenerative disease of the central nervous system that can infect humans and animals.The diagnosis of this disease is difficult,and currently depends mainly on PrP^Sc detection of brain tissue.Clinical brain biopsy is very traumatic and difficult for patients to accept,so most of the brain tissue sampling for the disease can only be performed after the patient's death.Compared with other countries,China's autopsy is difficult to implement due to culture difference.VPSPr is a novel human prion disease,its pathogenesis is not well understood.CWD,a highly infectious animal prion disease,can cause great harm to agriculture and environment.Therefore,it is very important to further understand various prion diseases and to find a readily accessible peripheral sample that can replace brain tissue for PrP^Sc detection.Researchers have observed the presence of infectious PrP^Sc in the skin through animal experiments,and RT-QuIC can be used to detect the seeding activity of PrP^Sc in the skin.Objective: To further understand the properties of VPSPr and CWD prion proteins.To verify the reliability of skin PrP^Sc as a biomarker for early diagnosis of prion disease;and determine the effectiveness of RT-QuIC in the detection of PrP^Sc from different prion diseases.Methods:(1)Establish RT-QuIC method and choose the optimal reaction conditions.(2)Detect PrP^Sc of the brain tissues of VPSPr and CWD in white-tailed deer by 1D and 2D Western Blotting(WB)as well as other techniques.(3)Determine brain PrP^Sc seeding activity of VPSPr and CWD deer by RT-QuIC,and compare the seeding activity of PrP^Sc in brain tissues between VPSPr and sCJD patients.(4)Use RT-QuIC to detect the seeding activity of skin PrP^Sc in 161 SCJD patients(almost all subtypes)and non-CJD patients,and analyze the specificity and sensitivity of this method for diagnosis of CJD,as well as the reliability of skin PrP^Sc as diagnostic specimens.Results:(1)The specific step-like electrophoresis pattern of VPSPr was dependent on the concentration of PK digestion and basic pH,and the PrP^Sc bands of VPSPr could only be detected by unique anti-PrP antibodies.The 1E4 antibody exhibits a higher affinity to the VPSPr-7 band.(2)Compared with sCJD PrP^Sc,the glycan with lower molecular weight in VPSPrPrP may have a smaller N-terminal or C-terminal truncation generating a PrP band that migrated slightly faster than that of full-length(FL)-PrP.(3)By RT-QuIC detection,the log SD50 per milligram of tissues were > 8.6 in sCJDMV2,>7.7 in GSS^P102L,> 7.1 in VPSPr129VV,7.0 in VPSPr129MM,6.1 in VPSPr129MV,and >5.7 in fCJD^V180I.PrP^Sc-seeding activity was lower in VPSPr brain than that in SCJD and GSS.(4)The PrP^Sc gel electrophoresis patterns of brain homogenates of different CWD isolates in white-tailed deer were not completely consistent,and could be divided into at least two types;(5)PrP^Sc-seeding activity was lower in the brain tissue of CWD than in sCJD,while the CWD PrP^Sc seeding-activity could still be detected at 10^-6 dilution.(6)The sensitivity and specificity of RT-QuIC for skin PrP^Sc detection in 161 patients with sCJD(almost all subtypes)and non-CJD patients were 88.8% and 100%,respectively.Conclusion:(1)The abnormal PrP glycosylation of VPSPr may be related to its pathogenesis;the detection sensitivity of its PrP^res was dependent on different antibodies,PK concentration and basis pH.(2)Different substrates had little effect on RT-QuIC results of the three VPSPr genotypes of PrP^Sc,and RT-QuIC could be used as a reliable diagnostic method for PrP^Sc detection;(3)Different CWD isolates may represent different PrP^Sc strains.RT-QuIC technology can be used as a reliable means of CWD screening and low amounts of PrP^Sc detection;(4)Skin PrP^Sc is a reliable biomarker for diagnosis of prion disease;(5)RT-QuIC is expected to be applied in the diagnosis of various prion diseases.