Development Of Nucleic Acid Testing Methods And Molecular Epidemiological Study Of Borna Disease Virus In Western China

BackgroundThe quick spreading of infectious viruses, including SARS in China,Niv in Malaysia, West nile virus in United States, and Avian Influenza Virus worldwide, have brought great panic to mankind. People started to reflect the reason why all those epidemics can't be predicted, monitored and stopped effectively. Early detection of potential outbreaks, exploration of origin, spreading, clinical manifestations- and- prognosis- associated host factors, diagnosis and clinic treatment, might be the best way to prevent epidemics.ObjectiveThe objective was to establish one-step real-time RT PCR, which is superior to traditional Fluorescent quantitative nested RT PCR, to rapidly detect Borna disease virus in diseased hosts, for nationwide molecular screening of potential infections. In addition, we also aim to perform molecular epidemiological study of BDV infection in brain tissues and blood samples of horses and swine in Xinjiang and Chongqing.Methods1. Based on GenBank target sequences, we designed TaqMan probes specifically targeting the conservative region of BDV p24 and established a one-step RT-PCR for detecting BDV infections. The sensitivity and specificity of established one-step RT-PCR were tested and compared with FQ-nRT-PCR.2. Since June 2007, a total of 480 samples collected from animal species in Western China were centrifuged to separate peripheral blood mononuclear cells (PBMCs). Total RNA was extracted by using Trizol agent. We have detected all the samples by using the optimized PCR assays. All positive samples were sequenced and further virus isolation was performed for epidemiological purpose.Results1. We logged on the NIH website (http://www.ncbi.nlm.nih.gov/BLAST/) to perform a BLAST search for the primers and probes. The designed primers and probes are matched for the published BDV sequences.2. The detection limit of the current one step RT-PCR was 1.7×102copies/μl,with a target range of 1.7×102～1.7×106copies/μl. The assay is highly specific and no cross-react with other virus sequences. The method can be properly applied to the BDV epidemiology research, with good sensitivity and specificity.3.We examined the PBMCs and brain tissues from horses and swine in Xinjiang and Chongqing for possible BDV infections. BDV was detected from both PBMCs and brain tissues in three horses (2.5%) in Yili, Xingjiang. The amplified positive products were > 98% homologous to standard He/80 strains. Of the 360 swine samples tested, BDV p24 RNA positive rate was 4.44% in Chongqing. The amplified positive products were 100% homologous to standard He/80 strains. Conclusion1. We have established one step RT-PCR and FQ-nRT-PCR for detection of BDV p24 RNA in diseased hosts. Compared with the one step RT-PCR, FQ-nRT-PCR is more sensitive and specific for epidemiological screening of potential BDV infections.2. The results of our epidemiological study suggest that there is BDV natural infection in horses in Yili, Xinjiang province. The BDV strains are highly homologous to He/80 standard strains.3. The results of our epidemiological study also suggest that there is BDV natural infection in swine in Three Gorges Regions, Chongqing. The BDV strains are highly homologous to He/80 standard strains.