Molecular Biological Study On Borna Disease Virus Infection In Neuropsychiatric Patients

Objective This study aimed to establish a fast and accuratequantitative method to detect Borna disease virus (BDV) ribonucleic acid(RNA) using fluorescence quantitative nested reverse transcriptasepolymerase chain reaction (FQ-nRT-PCR). It also intended to investigatethe correlation between BDV infection, some human neuropsychiatricdisorders and tumors of central nervous system, so as to provide earlywarnings for the potential epidemic outbreak of BDV infectious disease,and lay an experimental and theoretic basis for aetiological diagnosis,prevention and treatment of some human neuropsychiatric disorders aswell. Methods 1. According to the specific sequence of BDV p24 gene, the primers 8and the fluorescence probe were synthesized as designed. The fragmentsamplified by PCR were cloned into the pMD18-T vector. The positiverecombinant plasmid would be used as standard quantitative template tomake the standard curve for sample detection. 2. The BDV p24 fragments in peripheral blood mononuclear cells(PBMCs) were detected by FQ-nRT-PCR from 158 patients withneuropsychiatric disorders [including 60 cases with unknown causes ofviral encephalitis diagnosed clinically, 20 cases of multiple sclerosis, 15 ofGuillain-Barre syndrome (acute 12 and chronic 3, respectively), 12Parkinson's disease, 2 Alzheimer's disease, 31 schizophrenia, and 16 mood(or affective) disorder, respectively], and 112 healthy blood donors.Molecular cloning and sequence analysis were performed. 3. The FQ-nRT-PCR was used to detect the BDV p24 fragments from60 samples of human tumor tissues of central nervous system (including 18cases of meningioma, 15 glioma, 12 pituitary adenoma, 3hemangioblastoma, 2 acoustic nerve tumor, 2 cerebellar medulloblastoma,1 brain metastatic papilloma, 2 cerebral cyst, 2 neurinoma, and 3 spinalmeningioma, respectively), which were confirmed by histopathologydiagnosis, and 14 normal brain tissues. Results 1. The standard curve revealed the linear relationship between 9Ct (cycle threshold) and template concentration (r=0.998). TheFQ-nRT-PCR method for the detection of BDV p24 fragment wassuccessfully established. 2. The positive rate of the BDV p24 fragment from all samples was3.3%. Thereinto, the positive rate of the BDV p24 fragment inneuropsychiatric disorders was 5.7%, and negative results in multiplesclerosis, Alzheimer's disease, affective disorders, and normal controls.Compared with the control group, the positive rate of BDV p24 fragment inPBMCs from neuropsychiatric disorders was significantly high (P<0.05);the positive rate of BDV p24 fragment in the PBMCs in the unknowncauses of viral encephalitis (4.48%) was significantly high (P<0.05); thepositive rate of the BDV p24 fragment in the PBMCs in Guillain-Barresyndrome (6.67%) was high, but without any statistical significance(P>0.05), and among them, the acute Guillain-Barre syndrome wasnegative, and chronic Guillain-Barre syndrome (Chronic inflammatorydemyelinating polyneuropthy) (33.33%) was significantly high (P<0.05);the positive rate of the BDV p24 fragment in the PBMCs in Parkinson'sdisease (17.67%) was significantly high (P<0.05); the positive rate of theBDV p24 fragment in the PBMCs in schizophrenia (9.7%) wassignificantly high (P<0.05). 3. The sequence of the BDV p24 fragment from the patients with viralencephalitis was in conformity with that of the Parkinson's disease, as wellas schizophrenia, and its homogeneity corresponding to the sequence ofBDV standard strains was 95%-98%. It was present 3 situs consistencysilent mutation (nt 1658 T→C, nt 1667 A→G, nt 1670 C→T,withmutation rate 3%) compared with C6BV, 2 situs consistency silentmutation (nt 1673 C→T, nt 1676 T→C,with mutation rate 2%) comparedwith BDV/MDCK, and 4 situs consistency silent mutation (nt 1649 T→C,nt 16...