Molecular Epidemiology Of Borna Disease Virus

BACKGROUNDWHO announced that an outbreak of SARS involving 32 countries and regions over the world in 2003 led to 8 422 infected cases including 919 deaths, and the case fatality rate was nearly 11%. The outbreak of influenza A H5N1 of the bird flu virus in South and Southeast Asia in 2004 resulted in more than 100 million chichens killed and the virus even spread to humans to cause the infected patients dead. In April 2010, WHO has published that more than 213 countries and territories worldwide have reported laboratory confirmed cases of pandemic influenza A H1N1 2009, including over 17 000 deaths from the first case of influenza A H1N1 in Mexico in April 2009 to March 2010. As of March 31, 2010, 120 000 confirmed cases with influenza A H1N1 had been cumulatively reported by 31 provinces in China and the deaths were beyond 800 cases. The shocking EID (emerging infectious disease, EID) events have hugely impacted human health, economic development and social stability for the past 7 years, which make people produce more new views on active prevention and control of EID and perfection of related public health, and pay high attention to the pathogen of EID.OBJECTIVEThe purposes of this study are to establish sample database of animals infected by EID BDV in northwest of China,Ili,Xinjinang,to obtain epidemiological BDV data, to come to understand the epidemic status of different species of animals,and to analyze the possible phylogeny of BDV, which provide reliable data for making preplans of prevention and control of BDV outbreak and lay solid foundation for improving public health security system. At the same time, the cell model of BDV p40 viral protein transfected will be constructed to establish the research platform for BDV pathogenesis. The BDV p40 fluorescent quantitative PCR diagnosis kit will be further evaluated to provide a reliable means of detection of BDV epidemiological investigation and clinical research.METHODS1. In sample collection, according to the specificity of geographical origins and species diversities of the hosts infected by BDV, Ili region in Xinjiang was selected as the point of sampling collection; Ili horse, Xinjiang donkey and dogs of different breeds as sampling animals; peripheral blood and brain tissue of animals as sampling objects.2. In virus detection, the gene fragments of BDV p24 RNA were detected from PBMCs in 8 breeds of 150 dog's samples with using FQ-nRT-RT PCR. The possible false positives were excluded by determination of both BDV p40 RNA fragments and the plasmid standard of pMD19 BDV H1766 p24. The analysis was performed on genetic sequence, homologous comparison, amino acid sequence and phylogeny after BDV p24 positive products were validated.3. In phylogenetic analysis, the 19 nucleotide sequences of BDV p24 positive fragments of Ili horses, Xinjiang donkeys and Kazakh tobets (a Kazakh shepherd dog) had been detected with using FQ-nRT-RT PCR from PBMCs in 873 animals of 5 species and 8 breeds. The homologous similarity of nucleotide and amino acid sequences was analyzed by alignment of BDV standard strain He/80, strain V and H1766, and gene phylogenetic tree was reconstructed to analyze BDV phylogeny.4. In cell model construction, the cell of human oligodendrocyte strain (OL cell) was cultured, which had been established from human oligodendroglioma cell. OL cells were transfected by plasmid pEGFP-N1-BDV p40 with BDV p40 gene by liposome (lipofectamine- 2000). Having been repeatedly screened by G418, the OL cell turned to be a transfected one which may stably express the BDV p40 protein. The expression of the BDV p40 gene of plasmid in the OL cells was identified by the techniques of RT-PCR amplification and agarose gel electrophoresis while the expression of BDV p40 protein by the observation of green fluorescent protein expression, IHC and ELISA determination. The model of transfected type of OL cell which may stably expressed BDV p40 protein was established.5. Kit evaluation. The first, in the evaluation of thermal stability, the reagent in the kit was divided into volume units of one PCR amplification reaction, and then two units were randomly selected to get heat treatment by 25℃and 37℃in constant temperature water bath, respectively. The seven durations of heat treatment were chosen including 3 h, 6 h, 9 h, 12 h, 24 h, 48 h and 96 h. The plasmid standard of pMD19 BDV H1766 p24 in 6 concentration gradients was immediately amplified with using PCR after accomplishment of heat treatment in the corresponding temperature and time. PCR products were identified by agarose gel electrophoresis and the kit thermal stability was evaluated by comparison of the CT and R2 values of PCR reaction in different temperatures and time durations. The second, in the reliability evaluation of BDV detection, the BDV p24 gene fragment in the quantitative BDV (+) OL cell and in the pMD19 BDV H1766 p24 plasmid standard were selected to be amplified by PCR, respectively. The kit reliability was evaluated by comparison of consistency of concentration gradients detected from the two detective objects.RESULTS1. In sample collection, the samples in Ili region belonged to 12 different species and breeds, including Ili horse, Xinjiang donkey, Xinjiang Bactrian camel, Tianshan red deer, Kazakh Tobet, German shepherd, Carolina dog, Siberian husky, Pointer hound dog, Beagle dog, German Doberman dog and Pit bull terrier. The total numbers of sampling animals was 897 cases, of which Ili horses were 582, Xinjiang donkey 204, Xinjiang Bactrian camel 4, Tianshan red deer 1, Kazakh Tobet 95, German shepherd 28, Carolina dog 14, Siberian husky 8, Pointer hound dog 6, Beagle dog 4, German Doberman dog 3 and Pit Bull Terrier 2. The sampling kinds were 3 in all, including peripheral blood, brain tissue and whole brain. The sampling numbers contained 897 blood samples, 1 573 of brain tissue and 43 of whole brain. The blood samples included those of 528 Ili horses, 204 Xinjiang donkeys, 160 different breeds of dogs, 4 Xinjiang Bactrian camels and 1 Tianshan Red Deer. The brain tissue samples consisted of those of 1 165 Ili horses (233 samples from frontal, parietal, occipital, temporal lobes or midbrain for each), of 305 Xinjiang donkeys (61 samples from frontal, parietal, occipital, temporal lobes or midbrain for each), and of 103 dogs (right temporal lobe). The whole brain samples involved in those of 37 Ili horse and 6 Xinjiang donkeys. 2. In virus detection, BDV p24 RNA fragments were found out only in Kazakh Tobet in 8 breeds of 150 cases and their overall positive rate was 11.0% (10/91). Compared with the strain BDV He/80 from horse and that of BDV S6 from sheep in Germany, the homologous similarity of Kazakh Tobet was 99.2% and 95.7%, and that of amino acid was 100% and 89.3%, respectively. The kinship of Kazakh Tobet was close to He/80 and next to S6.3.In phylogenetic analysis, one part of nineteen nucleotide sequences formed Ili independent branch and the other clustered within Ili - Germany - Switzerland mixed branch. The independent branch did not cluster with standard strains and the homologous similarity of nucleotide sequences was 98% and that of amino acid 100% in independent branch. The German horse He/80 clustered into mixed branch and the homologous similarity of both nucleotide and amino acid sequences was 100% aligned by He/80 in mixed branch.4. In cell model construction, the conservative fragment of nucleotide sequence of BDV p40 gene in transfected type of OL cell was amplified by RT-PCR. The amplified product appeared a clear 154 bp band in agarose gel electrophoresis, which of the size was consistent with the target sequence. The expression of green fluorescent protein in OL nucleus was significantly greater than that in the cytoplasm (the gray value of nucleus / cytoplasm = 2.43) under the Inverted fluorescence microscope, which was fully consistent with biological nuclear localization of BDV p40 protein. The expression of BDV p40 protein was determined by polyclonal antibody with cell immunohistochemistry and CIC monoclonal antibody with ELISA in pEGFP-N1-BDV p40, pEGFP-N1 and normal OL cell. The results showed that there were negative in the former while all weak positives in the latter in which there was no significant difference in three kinds of cells.5. In the evaluation of thermal stability, there was no difference among the CT values of PCR reaction in 7 different heat treatment durations for each temperature after the kit got to heat treatment by 25℃and 37℃.The range of duration to keep normal PCR amplification efficiency was from 3 h to 96 h. There was no difference between the mean R2 value of two different temperatures (25℃and 37℃). In the reliability evaluation of BDV detection, the four concentration gradients (100-10-3) were detected by the kit in the concentration gradients (100-10-11) of quantitative BDV (+) OL cell and of pMD19 BDV H1766 p24 plasmid standard (100-10-5). The lowest detective concentration was 10-3.CONCLUSION 1. The sample size of the blood and brain tissue collected in the Ili region in different animals could basically reflect the status of BDV natural infection in the region. The sex and age of enrolled animals had good homogeneity which further improved reliability of the survey results. A new method of rapid craniotomy for the whole brain of equine was established for the first time at home and abroad.2. There may exist BDV natural infection in Ili region, and Kazakh Tobet was the reservoir host which positive rate of natural infection was 11.0% (10/91). BDV endemic strains in the region were concerned with the cross-infection of Ili horse with He/80 and the importing infection of Merino sheep with BDV S6 introduced from Saxony, Germany.3. It was first put forward that there could exist BDV independent strain due to geographical origin in host animals in Ili valley, Xinjiang. The reason that different kinds of hosts were infected by the same strain virus was the spread of foreign pathogen via the Prairie Silk Road.4. The transfected cell model of BDV p40-OL was established through human OL cells transfected by Plasmid pEGFP-N1- BDV p40. The cell model could provide an effective means for exploring the function of single viral protein in the host cell and the proteomics research for pathogenesis of depression caused by BDV. 5. The kit was of good thermal stability for it was not restricted by a certain range of temperature (25℃/ 37℃) and of sustainable time (3 h-96 h) at the temperature. It was of very high reliability of BDV detection for it could detect 10-3 BDV concentration the in OL cell.