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

肾综合征出血热病毒分子诊断的建立及其在无病毒动物培育中的应用

• 批准号: 01480512
• 负责人: ISEGAWA Yuji
• 金额: $ 4.1万
• 依托单位: Osaka University (1991)Kobe University (1989-1990)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480512/
• 关键词: Hemorrhagic Fever with Renal Syndrome Virus; Molecular Diagnosis; Virus Infected Animal; Virus Virulence; PCR Cloning; Dialect Sequencing; 賢症候性出血熱ウイルス

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

1)本文克隆了肾综合征出血热病毒B-1株S片段，用与M片段相同的方法测定了其全部核苷酸序列。将推导的氨基酸序列与同一血清型、汉滩株和Hallnas株的SR-11进行比较，同源性分别为98.4%、83.2%和61.7%。氨基酸序列相似性分析表明，B-1株由汉滩株进化而来，SR-11株由B-1株进化而来。此外，还发现在四种血清型中通常有两个位点的氨基酸序列发生变化。将B-1株L片段的氨基酸序列与汉滩株和Hallnas株的同源性分别为83.0%和73.5%，表明L片段在HFRS病毒的3个片段中具有最高的保守性。此外，预测编码依赖RNA的RNA聚合酶活性中心的区域高度保守。L段还演示了 ...更多信息 2）汉坦病毒属（Hantavirus）的汉坦病毒（Hantaan virus，HTN）在乳鼠腹腔内或脑内感染后引起致命性疾病。通过在Vero E6细胞中形成噬斑而从HTN的76-118菌株获得的HTN cl-1在小鼠中表现出增加的死亡率，而用HTN cl-2感染的小鼠没有显示出临床症状（Tamura等人，1989年）。作为理解毒力显著差异的分子基础的第一步，我们比较了HTN cl-1基因组的大（L）、中（M）和小（S）片段与HTN cl-2基因组的核苷酸序列，发现由M基因组片段编码的糖蛋白的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之间存在几个核苷酸和氨基酸差异，但76-118株糖蛋白的第1124位氨基酸也是与HTN cl-1相同的丝氨酸，HTN cl-1也是致病病毒。这些结果提示，这种替换可能是导致某些与毒力相关的病毒特性的原因。3）感染HFRS病毒的大鼠通常持续保持病毒状态，成为病毒携带者，血液中不显示病毒。然而，在一些大鼠中，可以在骨髓和白细胞中检测到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，该受试者在血液中显示出高抗体滴度而没有显示出任何病毒抗原。结果，可以分离出汉滩型病毒基因组，这表明该病毒即使在人类中也可以作为基因组长期存在。少

项目成果

ISEGAWA, Y., OHSHIMA, A., MURAKAMI, H., SOKAWA, Y.: "Detection of Hantaan virus RNA sequences by in vitro DNA amplification." Acta Virol.

ISEGAWA, Y.、OHSHIMA, A.、MURAKAMI, H.、SOKAWA, Y.：“通过体外 DNA 扩增检测汉滩病毒 RNA 序列。”

ISEGAWA, Y., UAMANISHI, K., UEDA, S.: "Association of serine in position 1124 of Hantaan virus glycoprotein with virulence in mice." Virology.

ISEGAWA, Y.、UAMANISHI, K.、UEDA, S.：“汉坦病毒糖蛋白 1124 位丝氨酸与小鼠毒力的关联。”

Y.Fujiwara: "The Preparation of cDNA Library and Direct Sequencing of Hemorrhagic Fiver with Rend Syndrome Virus Strain Biken 1 with Polymerase Chain Reaction"

Y.Fujiwara：“利用聚合酶链反应制备 Rend 综合征病毒 Biken 1 株出血性 Fiver 的 cDNA 文库和直接测序”

Y.Isegawa: "Detection of Hautaan virus RNA sequences by in vitro DNA anplification." Acta Virol.

Y.Isekawa：“通过体外 DNA 扩增检测 Hautaan 病毒 RNA 序列。”

Y.Fujiwara: "The Preparation of cDNA Library and Direct Sequencing of Hemorrhagic Fever with Renal Syndrome Virus Strain Bー1 with Polymerase Chain Reaction"

Y.Fujiwara：“肾综合征出血热病毒 Bー1 株 cDNA 文库的制备和聚合酶链反应直接测序”