Establishment of molecular diagnosis for hemorrhagic fever with renal syndrome virus and its application for the development of the virus free animals.

1)克隆了肾综合征出血热病毒B-1株S片段，其核苷酸序列测定方法与M片段相同。将推导的氨基酸序列与同一血清型的SR-11、汉滩株和哈尔纳斯株的氨基酸序列进行比较，同源性分别为98.4%、83.2%和61.7%。氨基酸相似性分析表明，B-1株是由汉滩株进化而来的，SR-11株是由B-1株进化而来的。此外，还发现在四个血清型中有两个常见的氨基酸序列改变的位点之一。将B-1株L片段的氨基酸序列与汉滩株和汉纳斯株的氨基酸序列进行比较，同源性分别为83.0%和73.5%，表明L片段在三个片段中具有最高的保守性。此外，预测的编码RNA依赖的RNA聚合酶活性中心的区域是高度保守的。L片段还演示了…2)汉坦病毒属的汉滩病毒(Hantaan Virus，HTN)可引起乳鼠脑内感染致死性疾病。从76-118株HTN通过在Vero E6细胞中形成菌斑而获得的HTN CL-1，在小鼠中显示出更高的死亡率，而感染HTN CL-2的小鼠没有临床症状(Tamura等人，1989)。作为了解毒力差异的分子基础的第一步，我们比较了HTN CL-1和HTN CL-2基因组大(L)、中(M)和小(S)片段的核苷酸序列，发现M基因组片段编码的糖蛋白在1124位只有一个替换，其中HTN CL-1的丝氨酸被HTN CL-2的甘氨酸取代。虽然亲本株与HTN CL-1株在核苷酸和氨基酸上存在一定的差异，但76-118株的糖蛋白1124位氨基酸与HTN CL-1株的丝氨酸相同，也是致病病毒。这些提示这种替换可能与某些与毒力相关的病毒特征有关。3)感染HFRS病毒的大鼠通常会持续保持病毒，并成为病毒携带者，血液中不显示病毒。然而，在一些大鼠的骨髓和白细胞中可以检测到依赖RNA的RNA聚合酶。因此，在本研究中，我们试图从49年前感染汉坦病毒的患者中分离出汉滩病毒的病毒基因，该患者血液中没有显示任何病毒抗原，但抗体效价很高。因此，可以分离到汉滩型病毒的基因组，这表明该病毒即使作为基因组在人类体内也可以长期保持。较少

1) S segment of B-1 strain of HFRS virus was cloned and all of its nucleotide sequence was determined by the same method as used for M segment. When the deduced amino acid sequence was compared to that of SR-11, which included in the same serotype, Hantaan and Hallnas strains, 98.4%, 83.2% and 61.7% homologies were observed, respectively. The amino acid similarities indicate that B-1 strain evolved from Hantaan strain and SR-11 strain came from B-1 strain by flame sift. Moreover, it was shown that there were one of two sites where amino acid sequence change observed in the four serotypes commonly. When the amino acid sequence of L segment of B-1 strain was compared to that of Hantaan and Hallnas strains, 83.0% and 73.5% homologies were observed, respectively, indicating that the L segment has the highest conservation in the three segments of HFRS virus. In addition, the region predicted to encodeactive center of RNA dependent RNA polymerase was highly conserved. The L segment also demo … More nstrated to have sites where amino acid sequence change observed in the four serotypes commonly.2) The Hantaan virus (HTN) of Hantavirus genus causes a fatal disease in suckling mice following intraperitoneal of intracerebral infection. HTN cl-1, which was obtained from the 76-118 strain of HTN by plaquing in Vero E6 cells, exhibited increased mortality rates in mice whereas mice infected with HTN cl-2 showed no clinical signs (Tamura et al., 1989). As a first step to understand the molecular basis for the marked difference in virulence, we compared the nucleotide sequences of the large (L), medium (M) and small (S) segments of HTN cl-1 genome with those of HTN cl-2 and found that there was only one substitution in position 1124 of the glycoprotein encoded by the M genome segment, in which serine of HTN cl-1 was replaced by glycine of HTN cl-2. Although there were several nucleotide and amino acid differences between the parental strain and HTN cl-1, the amino acid in position 1124 of the glycoprotein of the 76-118 strain was also the same serine as HTN cl-1, which was also pathogenic virus. These suggest that this substitution may be responsible for some of the virus characteristics related to virulence.3) The rats infected with HFRS virus usually maintain the virus persistently and become virus carrier without showing viruses in the blood. However, in some rats RNA dependent RNA polymerase can be detected in the bone marrow and leukocytes. Therefore, in the present study, we tried to isolate virus genes of Hantaan type virus from a subject who had been infected with Hantavirus 49 years ago demonstrating high antibody titer without showing any virus antigens in the blood. As a results, virus genome of Hantaan type could be isolated, and thus indicate that the virus can be maintained for a long period even in human as genome. Less