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Detection of Rna and Phylogenetic Analysis of Borna Disease Virus in Multi-species in Xinjiang and Chongqing, China

Author: ZhangYingYing
Tutor: XiePeng
School: Chongqing Medical University
Course: Neurology
Keywords: Borna disease virus Fluorescence quantitative nested reverse transcription polymerase chain reaction p24 fragment p40 fragment Phylogenetic
CLC: R181.3
Type: PhD thesis
Year: 2010
Downloads: 19
Quote: 0
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Abstract


Background Borna disease virus (Borna disease virus, BDV) is a new-onset central nervous system infection virus, it is a segment enveloped single-stranded negative-strand RNA viruses, with the the fibrils virus Branch, vice myxovirus Branch and rhabdovirus Branch attributable to common single strand of negative-strand RNA viruses head (Monnegaviradae), and the case of the ad hoc the Borna virus Division Borna virus for the virus. 1895 BDV (Borna) town in Germany Borna horses caused the outbreak, hence the name. Date, BDV host in nature, worldwide distribution and transmission routes continue to have a new discovery. Initially thought the the BDV infection only in Germany and Switzerland, the horses and the sheep, occasionally sporadic cases. In recent years, with the emergence of more BDV diagnostic techniques and the depth of the world BDV research, known infected animal species has increased year by year. Under natural conditions, including chickens, goats, rabbits, donkeys, llamas, alpacas, cattle, dogs, cats, and ostriches, including a variety of warm-blooded animals that could be BDV infection. Previous studies have shown that populations with animals the BDV infection throughout Europe, Asia and the Americas, to estimate its actual geographical distribution may be more widely. The route of transmission of the virus is still unclear, known study found that the virus could infect animals with respiratory, conjunctival discharge and fecal secretions, and spread the infection to other animals and humans through the nose. In China, Hagiwara K, Zhu Dan, Ai-Ying Yang, Xu Ping, Zhao Libo Liu Qingjun, Wang Zhenhai and Chen CH have reported in Xinjiang, Heilongjiang, Chongqing, Ningxia, Guizhou and Taiwan people, horses and sheep naturally infected with BDV, animals and humans in China there the BDV's natural infection, but in different time stages of animal and human BDV infection and spread natural sources, route of transmission is unclear. The aim of the present study was designed to pre-experiment based on the support of the National High Technology Development Program (863 Program, 2006AA02Z196) to study Chinese in Xinjiang and Chongqing region multi-species the BDV natural infection, and analyze their phylogenetic sources for tracing source of infection, the spread between the analysis of the geographic distribution of BDV and elucidating intraspecies and provide new ideas and the basis for the etiology of neuropsychiatric disease diagnosis and its control. Method 1. Early development of Borna disease virus fluorescence quantitative PCR diagnostic kit using fluorescence quantitative nested reverse transcription polymerase chain reaction (Fluorescence quantitative nested transcription polymerase chain reaction, FQ-nRT-PCR) detection of Xinjiang Ili region stocking 518 the horses health Ili horses and of 200 healthy Xinjiang donkey in peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) BDV p24 fragment using the the the ordinary reverse transcriptase polymerase chain reaction (Reverse transcription polymerase chain reaction, RT-PCR) to detect BDV p40 gene fragments of the same samples deeper verification. Used in this experiment BDV p24 fluorescence in standard quantitative PCR diagnostic kit in order to exclude the possibility of the plasmid pollution for PMD19 plasmid, on all samples at the same time detection of PMD19 plasmids. Ili horse BDV p24 fragment sequences detected positive samples into the NCBI Blast with five countries in the GenBank seven animal species are 33 cases BDV p24 gene sequence and pre Xinjiang region BDV p24 fragment positive cases of BDV p24 gene sequences than analysis of nucleotide and amino acid sequence homology, the reconstruction of gene phylogenetic tree analysis Ili horses and Xinjiang donkey in BDV possible sources and routes of transmission. 2 FQ-nRT-PCR detection the Chongqing region healthy cattle, goats and pigs, 50 patients in PBMCs BDV p24 fragment, deeper verification using ordinary RT-PCR to detect BDV p40 gene fragments of the same sample. In addition, in order to exclude the possibility of the plasmid pollution, all samples PMD19 plasmid detection. BDV p24 fragment sequences detected positive samples into the NCBI Blast seven animal species and five countries in GenBank are 33 cases BDV p24 gene sequence and Chongqing in the Early BDV p24 fragment positive domestic chickens, goats, dogs, pigeons, house rabbits and pigs, six cases of BDV p24 gene sequence comparison analysis of nucleotide and amino acid sequence homology, to rebuild gene phylogenetic tree, comparing different time stages BDV natural infection with or without changes; different species at different times. different stages of germ-line sources or without, so as to lay the foundation to further explore the the BDV natural source of infection and the route of transmission. 3. FQ-nRT-PCR to detect 20 cases of patients with viral encephalitis (Viral encephalitis, VE), patients with central nervous system infection, 52 cases and 18 cases of healthy donors PBMCs BDV p24 fragment, using ordinary RT-PCR to detect BDV p40 gene fragments of the same samples deeper verification. In addition, in order to exclude the possibility of the plasmid pollution, all samples PMD19 plasmid detection. Detected positive samples of BDV p24 fragment the sequence entering NCBI carried Blast are 33 cases of BDV p24 gene sequence and Chongqing in the Early BDV p24 fragment positive schizophrenia, Parkinson's disease (Parkinson's disease, seven animal species and five countries in the GenBank PD), four cases of Guillain-Barre syndrome (Guillain-Barr├ęsyndrome, GBS) and VE patients BDV p24 gene sequence sequence and phylogenetic analysis, analysis of the nucleotide and amino acid sequence homology, reconstruction gene phylogenetic tree, compare natural infection rates in different time stages with or without changes; compare whether the different time periods of different diseases germline sources so as to lay the foundation to further explore the the BDV natural source of infection and the route of transmission. Results 1. FQ-nRT-PCR detection of Xinjiang Yili Prefecture animal samples RNA BDV p24 gene fragment. Found that nine cases samples BDV p24 testing positive (Ili horse: 5/518, 0.97%; Xinjiang donkey: 4/200, 2.00%); detected the BDV p40 fragments was positive in 9 cases, with BDV p24 positive results, and the rest of the samples were negative; in BDV p24 and p40 test results are positive samples were not detected in the plasmid standard, all samples tested were not found in the standard pollution; nine cases of positive samples BDV p24 gene fragment sequences are identical, with the BDV standard virus strains Strain V, H1766 and He/80 Geometric nine cases of positive samples nucleotide and amino acid homology similarity of 94% to 100% and from 82% to 100%, respectively, which with He/80 strains homologous up to 100%; Ili horse three cases, pre-Xinjiang region BDV p24 gene sequence comparison, nine cases of positive samples nucleotide and amino acid homology similarity of 99% to 100% and from 96% to 100%, respectively; this detected nine cases of positive samples and pre-Ili horse three cases of positive samples BDV p24 fragment sequences converge Germany - Switzerland - Austria - Japan - China's Xinjiang mixed lineage. 2. FQ-nRT-PCR detection of Chongqing animal samples RNA BDV p24 gene fragment. The results found that testing positive (cattle: 3/50, 6.00%; sheep: 2/50, 4.00%; pigs: 2/50, 4.00%); seven cases detected BDV p40 fragment positive specimens BDV p24 and BDV the p24 positive results, the remaining samples were negative; BDV p24 and p40 test results were positive samples were not detected in the plasmid standard, all samples tested were not found in the standard pollution; This experiment detected seven cases of cattle goats and pigs positive sample nucleotide sequence and amino acid sequences are identical, with the the BDV standards the virus isolates Strain V the H1766 and He/80 geometric seven cases of positive samples nucleotide and amino acid homology similarity were 94% to 100% and 82% to 100%; six cases of pre-Chongqing region home chickens, goats, dogs, pigeons, rabbits and pigs BDV p24 gene sequence alignment, Homology 94% to 100% and 82%, respectively, to 100%; detected seven cases of positive samples BDV p24 fragment sequences and pre-BDV p24 fragment sequence detection Chongqing area pig convergence Germany - Switzerland - Austria - Japan - China Chongqing mixed lineage, early detection home chickens, goats, dogs, pigeons, rabbits positive samples BDV p24 gene fragment sequences form an independent branch of Chongqing, China. FQ-nRT-PCR detection of the Chongqing area sample RNA BDV p24 gene fragment. The results found that the five specimens the BDV p24 testing positive (VE patients: 3/20, 15.00%; cerebrovascular disease (Cerebrovascular disease, CVD) patients: 2/32, 6.25%; GBS patients, patients with epilepsy, multiple sclerosis (Multiple sclerosis, MS) patients, PD patients, alcoholic encephalopathy and healthy blood donors were negative); detected BDV p40 fragment was positive in five cases, with BDV p24 positive results, the remaining samples were negative; BDV p24 and p40 detection the plasmid standard results are positive samples were not detected in all samples tested were not found in the standard pollution; BDV standard virus strains Strain V H1766 and He/80 geometric, the experimental detection of patients with two cases of VE 94% to 100% and 82% and 100%, respectively, and two cases of CVD patient samples nucleotide and amino acid homology similarity; 1 cases VE of 95% to 98% and from 89% to 93% of patients; Chongqing in the Early BDV p24 gene sequences in schizophrenia, PD, GBS and VE 4 patients, 2 cases of VE patients, and two cases of CVD patient samples nucleotide and amino acid homology similarity of 94% to 97% and 82%, respectively, to 89 %, 1 case of VE patients from 95% to 100% and from 93% to 100%; detected four cases of positive samples (two cases of CVD patients and two cases the VE of patients) the BDV p24 fragment the sequence convergence Germany - Switzerland - Austria - Japan - China Chongqing mixed lineage, another one cases the VE of patients positive samples and early detection of schizophrenia, PD, GBS and VE patient samples BDV p24 gene fragment sequences form an independent branch of Chongqing, China. Conclusion 1. Ili horse, health of China's Xinjiang region and Xinjiang donkey there are the BDV natural infection. Based on phylogenetic analysis of BDV infection may be caused by the virus transfers in Germany, Switzerland, Austria, Japan and China, countries. Rebuild germline occurred inside the tree branches, BDV there is a high degree of homology cycle spread between the animal and the animal that may exist between the different host donkey in of Yili horse and Xinjiang. Chongqing, China healthy cattle, goats and pigs, there are infection of BDV natural. Chongqing area animal hosts may exist based on phylogenetic analysis, the the endogenous the BDV independent strains originating from outside of Phytophthora incoming epidemic strains. The BDV germline may vary in different species and period can be expressed as a particular system mainly illustrate the BDV its host may exist evolution and variation of stable equilibrium. VE patients, and patients with CVD in Chongqing, China, there are infection of BDV natural. Chongqing region based on phylogenetic analysis, there may be people host the endogenous the BDV independent strains and epidemic strains originated from outside of Phytophthora incoming, the conclusion Chongqing area animal, animals and animal, the animal may exist between different host The cycle between human beings and human communication and human.

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