宁夏地区病毒性脑炎中新发博尔纳病病毒感染的研究

Study on Borna disease virus infection in viral encephalitis patients in Ningxia Hui Autonomous Region

目的 研究宁夏地区新发博尔纳病病毒（BDV）在病毒性脑炎患者中的感染状况,分析病毒株的核酸序列、编码的氨基酸序列和病毒株的系统发生.方法 套式反转录实时荧光定量PCR检测60例病毒性脑炎患者血液标本;并对前期课题组检测的59例病毒性脑炎患者中BDV p24阳性样本和本实验PCR检测阳性标本使用ELISA法检测其对应脑脊液中p40抗体,对阳性标本进行连接测序,应用MEGA和DnaSP4.0软件对测序结果与国外标准株He/80、H1766、strain V等的核酸和氨基酸序列对比分析,构建病毒基因的系统发生树.结果 PCR检测119份血标本,发现有12份标本BDV p24阳性,阳性检出率为10.08%,7份BDV p40检测阳性,阳性率为5.88%;ELISA检测脑脊液核蛋白抗体,有2份阳性,阳性检出率为1.68%.基因聚合分析显示,7份BDV阳性的核酸序列中有6份与德国马源性的He/80株同源性达100%,1份仅有1个碱基发生同义突变;转录后的氨基酸对比分析显示,7份与He/80株完全一致;重构的基因系统发生树中可见宁夏病毒性脑炎患者BDV核苷酸序列与国外标准株形成一个中国（宁夏）-德国-日本的混合支系.结论 宁夏地区病毒性脑炎患者中可能存在BDV感染,其发生可能与动物的密切接触相关,病毒在种系发生中与德国马源性He/80株有极高的同源性,推断病毒有可能从国外传入本土,不排除病毒株变异的可能.

Objective To investigate the prevalence of Borna disease virus （BDV） in viral encephalitis patients in Ningxia Hui Autonomous Region and to analyze phylogenetic development of BDV in this area. Methods The gene fragments of BDV were amplified by real time nested reverse transcriptase polymerase chain reaction （PCR） from peripheral blood mononuclear cells （PBMC） of 60viral encephalitis （VE） patients. Enzyme linked immunosorbent assay （ELISA） was applied to detect p40 antibody in the cerebrospinal fluid from those patients who were confirmed as BDV p24 positive in previous study and those patients who were detected as BDV positive by PCR in the present study.The confirmed p40 positive samples were used for BDV gene cloning and sequencing, which were subsequently compared with the sequences of overseas standard strains, including He/80, H1766 and strain V by software MEGA and DnaSP4.0. The virus phylogenetic tree was constructed based on the above data. Results The positive rate of BDV p24 by PCR was 10. 08% （12/119） and the positive rate of BDV p40 by PCR was 5.88% （7/119）. The cerebrospinal samples from two patients were detected as nucleoprotein antibody positive by ELISA, which resulted in a positive rate of 1.68%. The results of gene sequence analysis showed that the sequences of 6 cases among the 7 BDV positive patients were exactly same as the sequence of German horse-origin He/80 strain. Only one synonymous mutation was detected in the remaining one case. Reconstructed gene phylogenetic tree showed a hybrid China （Ningxia）- German and Japan branch was formed by the BDV stain isolated from VE patients in Ningxia and the overseas BDV standard stains. Conclusions BDV infection may exist in VE patients in Ningxia Hui Autonomous Region, which may be associated with close contact between human and animals. The gene sequence of stains isolated from patients in Ningxia is highly homological with the overseas standard strain, which suggests that the domestic BDV may come from overseas, while domestic strains with mutations couldn＇t be excluded.