| BackgroundOf late, Borna disease virus (BDV) is one more agent added to the list and is under close scrutiny as the causative agent of neuropsychiatric disorders. BDV is an enveloped, non-cytolytic, non-segmented negative strand RNA virus, belonging to family Bornaviradae of the order Monnegaviradae. The other viruses in the Monnegaviradae group are Filoviradae, Paramyxoviradae and Rhabdoviradae. BDV is the causative agent of Borna disease (BD), a fatal non-purulent meningoencephalitis that was originally described in Germany and Switzerland. It is named after the town Borna in Sexonomy/Germany, where an epidemic attack of BD occurred in cavalry horses 100 years ago.More and more is known regarding the epidemiology of BDV. Originally, it was supposed to be a natural pathogen of horses and sheep in South Eastern Germany, but now it is known it may infect several species ranging from birds to primates across the world. Its transmission is unclear. The virus may be shed with secretions from the respiratory tract, conjunctiva and faeces of infected animals and is supposed to spread intranasally. BDV has a particular predilection for the neurons of the limbic system, basal ganglia and the brain stem and diffuses throughout the CNS. It replicates locally in the neurons and migrates intra-axonally and spreads by direct cell to cell contact via neuroreceptors in the olfactory epithelium to the adjacent brain tissue.In China, scattered reports demonstrated natural infection of BDV in horses, sheep and human in Xinjiang, Heilongjiang, Chongqing, Ningxia, Guizhou and Taiwan, which shows the existence of BDV infection in animals and human in China. But the animals and human BDV infection in different periods and the natural sources is not clear.ObjectiveThe objective of this study is to investigate the epidemiology of BDV infection in multi-species and gain detailed insights into the genetic characters and phylogeny of BDV in Xinjiang and Chongqing, China.Methods1. BDV p24 fragment was detected by fluorescence quantitative nested transcription polymerase chain reaction from peripheral blood mononuclear cells of 518 healthy Yili horses and 200 healthy Xinjiang donkeys in Yili, Xinjiang. Positive samples were confirmed by detecting BDV p40 fragment and plasmid to preclude the contamination. By alignment with thirty-three BDV p24 sequences of seven animals in five countries from GeneBank and three positive gene sequences of the Yili horses in preliminary studies in Xinjiang, the sequence homologous similarity of the nucleotide and the amino acid sequences of BDV p24 positive samples was analyzed and gene phylogenetic tree was reconstructed.2. BDV p24 fragment was detected by fluorescence quantitative nested transcription polymerase chain reaction from peripheral blood mononuclear cells of fifty healthy cattle, fifty healthy goats, fifty healthy pigs in Chongqing. Positive samples were confirmed by detecting BDV p40 fragment and plasmid to preclude the contamination. Phylogenetic analysis of BDV p24 positive samples were performed by alignment with six positive gene sequences of the chicken, goat, dog, pigeon, rabbit and pig in preliminary studies in Chongqing and thirty-three sequences of seven animals in five countries from GeneBank. The sequence homologous similarity of the nucleotide and the amino acid sequences was analyzed and gene phylogenetic tree was reconstructed.3. BDV p24 fragment was detected by fluorescence quantitative nested transcription polymerase chain reaction from peripheral blood mononuclear cells of twenty patients with viral encephalitis, fifty-two non-central nervous system infection patients and eighteen blood donors in Chongqing. Positive samples were confirmed by detecting BDV p40 fragment and plasmid to preclude the contamination. Phylogenetic analysis of BDV p24 positive samples were performed by alignment with four positive gene sequences of patients with Schizophrenia, Parkinson's disease, Guillain-Barrésyndrome and viral encephalitis in preliminary studies in Chongqing and thirty-three sequences of seven animals in five countries from GeneBank. The sequence homologous similarity of the nucleotide and the amino acid sequences was analyzed and gene phylogenetic tree was reconstructed.Results1. The positive rates of BDV infection in PBMCs of 518 healthy Yili horses and 200 healthy XinJiang donkeys in Xinjiang were 0.97% and 2.00%, respectively. The results of BDV p40 detection confirmed all of the samples with BDV p24 positive. All the samples tested were not contaminated by plasmid. Aligned with Strain V, H1766 and He/80, the homologous similarity in nine sequences of both the nucleotide and the amino acid was from 94% to 100% and 82% to 100%. And aligned with the three positive gene sequences of the Yili horses in preliminary studies, the homologous similarity in nine sequences of both the nucleotide and the amino acid was from 99% to 100% and 96% to 100%. The nine nucleotide sequences of BDV p24 fragment in the positive samples and the three positive gene sequences of the Yili horses in preliminary studies clustered within Germany - Switzerland - Austria - Japan - Xinjiang mixed branch.2. The positive rates of BDV infection in PBMCs of fifty healthy cattle, fifty healthy goats, fifty healthy pigs in Chongqing were 6.00%, 4.00% and 4.00%, respectively. The results of BDV p40 detection confirmed all of the samples with BDV p24 positive. All the samples tested were not contaminated by plasmid. Aligned with Strain V, H1766 and He/80, the homologous similarity in seven sequences of both the nucleotide and the amino acid was from 94% to 100% and 82% to 100%. And aligned with the six positive gene sequences of the chicken, goat, dog, pigeon, rabbit and pig in preliminary studies, the homologous similarity in seven sequences of both the nucleotide and the amino acid was from 94% to 100% and 82% to 100%, too. The seven nucleotide sequences of BDV p24 fragment in the positive samples and the positive gene sequence of the pig in preliminary studies in Chongqing clustered within Germany - Switzerland - Austria - Japan - Chongqing mixed branch. And the five positive gene sequences of the chicken, goat, dog, pigeon and rabbit in preliminary studies in Chongqing formed Chongqing independent branch.3. The positive rates of BDV infection in PBMCs of twenty patients with viral encephalitis, thirty-two patients with cerebrovascular disease in Chongqing were 15.00% and 6.25%, respectively. The results of BDV p40 detection confirmed all of the samples with BDV p24 positive. All the samples tested were not contaminated by plasmid. Aligned with Strain V, H1766 and He/80, the homologous similarity in four sequences of both the nucleotide and the amino acid was from 94% to 100% and 82% to 100%, and in the one was 95% to 98% and 89% to 93%. And aligned with the four positive gene sequences of patients with Schizophrenia, Parkinson's disease, Guillain-Barrésyndrome and viral encephalitis in preliminary studies in Chongqing, the homologous similarity in four sequences of both the nucleotide and the amino acid was from 94% to 97% and 82% to 89%, and in the one was 95% to 100% and 93% to 100%. Among the five nucleotide sequences, one of the patient with viral encephalitis formed Chongqing independent branch and the other clustered within Germany - Switzerland - Austria - Japan - Chongqing mixed branch. And the four positive gene sequences of patients with Schizophrenia, Parkinson's disease, Guillain-Barrésyndrome and viral encephalitis in preliminary studies in Chongqing formed Chongqing independent branch, too.Conclusion1. Our study suggested BDV natural infection in Yili horses and Xinjiang donkeys in Xinjiang. According to the phylogeny, the epidemic strain might due to spread of foreign pathogen. And it may exist animal-to-animal transmission by contact of host animals.2. Our study suggested BDV natural infection in healthy cattle, goats, and pigs in Chongqing. According to the phylogeny, there may exist the Chongqing independent BDV strain from geographical origin,and the epidemic strain due to spread of foreign pathogen.3. Our study suggested BDV natural infection in patients with viral encephalitis and cerebrovascular disease in Chongqing. According to the phylogeny, there may exist the Chongqing independent BDV strain from geographical origin,and the epidemic strain due to spread of foreign pathogen. And it may exist transmission between animals, between animals and people, and between people.
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