博尔纳病毒在宁夏的人和动物感染的检测

Detection of Borna virus infection in human and animal in Ningxia

目的对临床VE患者以及其本人接触动物的检测,探讨BDV在宁夏地区的感染状况及感染机制,并分析BDV核酸和转化后的氨基酸序列,构建病毒种系发生的来源。方法应用巢式逆转录酶聚合酶链反应结合荧光定量PCR检测患者及其接触的动物的外周血单核细胞的p24和p40基因片段。对检测阳性标本进行连接测序,应用MEGA和DnaSP4.0软件对测序结果同国外的标准株HE80、H1766、strainV等进行核酸和氨基酸序列对比分析,并构建病毒基因的系统发生树。结果在60例VE患者及其接触的710绵羊中,PCR检测发现有3例阳性,阳性检出率5%;9头绵羊阳性,阳性检出率为1.27%。基因聚合分析显示人和动物BDV的核酸序列和编码氨基酸序列与德国马源性的HE80株同源性高达100%,重构基因系统发生树可见宁夏VE患者和牛羊BDV核苷酸序列与国外的标准株形成宁夏-德国-日本的混合支系,同时宁夏绵羊BDV序列也形成了一个独立的支系。结论宁夏地区人和牛羊存在BDV的自然感染,并且人的感染存在动物源性,其传染途径很可能是呼吸道的传播可能引起脑炎的非特异性的临床症状,病毒在种系发生中与德国马源性HE80株有极高的同源性,病毒可能由国外引入本土,不排除病毒株变异的可能,人和绵羊之间存在相互传染的可能。

The purpose of this study was to investigate the infection of Borna disease virus （BDV） in viral encephalitis （VE） patients and the animals they exposed to, and to probe BDV infection mechanisms in Ningxia region. BDV nucleic acid and the amino acid sequences were analyzed after transformation, phylogenetic origin of the virus was constructed as well. The P24 and P40 fragments of BDV were detected by nested reverse transcriptase polymerase chain reaction （RT-PCR） with real time PCR in peripheral blood mononuclear cells （PBMCs） from VE patients and the exposed animals. Before comparing with the overseas strains including HE80, H1766 and strain V by MEGA and DnaSP4.0, the positive samples were cloned and sequenced to construct the phylogenetic tree. A total of 3 examples of BDV were detected to be positive in 60 VE patients. The positive rates of BDV for cattle sheep were 1.27%（9/710）. The gene cluster analysis showed that the nucleotide sequence and deduced amino acid sequence of human and animal BDV were 100% homologous with the horse-derived strain HE80 in Germany. The reconstruction of the gene phylogenetic tree showed that the BDV nucleotide sequences of VE patients and animals in Ningxia have form Ningxia-Germany-Japan hybrid branch with oversea standard strains. At the same time, the BDV sequences of sheep in Ningxia have also form an independent branch. There was a natural infection of BDV among human and animal in Ningxia Province and the infection source was probably originated from animals. Respiratory tract was likely to be the main channel of infection and may cause non-specific clinical symptoms such as encephalitis. The gene sequence was highly homologous with HE80 detected from ill horses in Germany. Results indicated that the virus might be introduced from abroad and have experienced gene mutation. There was also a possibility in cross-infection among human, and sheep.