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HFRS病毒B-1株S片段，采用与M片段相同的方法测定其所有核苷酸序列。将所得氨基酸序列与同血清型的汉坦株和哈尔纳株SR-11进行比较，同源性分别为98.4%、83.2%和61.7%。氨基酸相似性表明B-1菌株由汉滩菌株演化而来，SR-11菌株由B-1菌株演化而来。此外，四种血清型中氨基酸序列变化普遍存在两个位点之一。将B-1株的L段氨基酸序列与Hantaan株和Hallnas株进行比较，同源性分别为83.0%和73.5%，说明L段在HFRS病毒3个片段中保守性最高。此外，预测的RNA依赖RNA聚合酶编码活性中心区域高度保守。L段也显示出4种血清型中常见的氨基酸序列变化位点。2)汉坦病毒属汉坦病毒（HTN）在哺乳小鼠脑内腹腔感染后可致死性疾病。htncl -1是通过在Vero E6细胞中斑块化从htn76 -118株中获得的，在小鼠中显示出死亡率增加，而htncl -2感染的小鼠没有表现出临床症状（Tamura等，1989）。作为了解毒力显著差异的分子基础的第一步，我们比较了HTN cl-1基因组大(L)、中(M)和小(S)片段与HTN cl-2的核苷酸序列，发现M基因组片段编码的糖蛋白只有1124位有一个替换，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htncl -1存在若干核苷酸和氨基酸差异，但76-118株的糖蛋白1124位氨基酸与htncl -1相同，也是致病性病毒。这些表明，这种替代可能是与毒力相关的一些病毒特征的原因。3)感染HFRS病毒的大鼠通常持续保持病毒，成为病毒携带者，但在血液中不显示病毒。然而，在一些大鼠的骨髓和白细胞中可以检测到RNA依赖的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受试者中分离汉坦型病毒的病毒基因，该受试者的血液中没有显示任何病毒抗原，抗体效价高。结果表明，汉滩型病毒基因组可以被分离出来，从而表明该病毒即使在人类基因组中也可以长期存在。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