Analysis of factors determining the host range of calicviruses

杯状病毒宿主范围决定因素分析

• 批准号: 11660299
• 负责人: TOHYA Yukinobu
• 金额: $ 2.3万
• 依托单位: THE UNIVERSITY OF TOKYO (2000-2001)Kagoshima University (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660299/
• 关键词: calicivirus; host specificity; receptor; dog; cat; capsid; non-structural protein; genome; 中和エピトープ; モノクローナル抗体; プロテアーゼ

The final goal of this study is to find viral and cellular factors that determine the host specificity of calicivirus by the use of feline and canine caliciviruses in cell culture systems. In this study, we obtained several new findings as follows.1. Molecular biological analysis of canine calicivirus (CaCV) : The complete nucleotide sequence of CaCV genome was determined. Two conformational neutralizing epitopes of CaCV identified by monoclonal antibodies (MAbs) were located in regions corresponding to the 5' and 3' hypervariable regions of the capsid protein of feline calicivirus (FCV).2. In vitro host ranges of FCV and CaCV : CaCV can only replicate in cell lines of canine organ. FCV can grow well in feline cell lines and some strains of FCV can replicate in Vero cells of simian origin. However, progeny viruses were equally produced in any cell lines by transfection of the RNA genomes from FCV and CaCV, suggesting that early interaction of the caliciviruses with cells may be the major determinant for their cell tropism in vitro.3. Virus-binding assay : Using a MAb produced in section 1., a virus binding assay by flow cytemetry was developed for CaCV. The analysis indicated that CaCV binds efficiently to the permissive cells, but does not bind to non-permissive cells.4. A MAb against MDCK cells of canine origin that blocks CaCV infection was produced and characterized. The MAb bound to the permissive cells but not to the non-permissive cells for CaCV and inhibited the binding of CaCV to the permissive cells, indicating that the MAb recognizes the receptor for CaCV. By immunoprecipitation, the MAb was shown to react with two proteins with molecular masses of 76 and 65 kDa.

本研究的最终目标是通过在细胞培养系统中使用猫和犬杯状病毒来找到决定杯状病毒宿主特异性的病毒和细胞因子。本研究得到以下几个新发现： 1．犬杯状病毒(CaCV)的分子生物学分析：确定了CaCV基因组的完整核苷酸序列。单克隆抗体(MAb)鉴定出CaCV的两个构象中和表位，位于猫杯状病毒(FCV)衣壳蛋白5'和3'高变区对应的区域。2． FCV和CaCV的体外宿主范围：CaCV只能在犬器官细胞系中复制。 FCV 可以在猫科动物细胞系中生长良好，并且某些 FCV 菌株可以在源自猿猴的 Vero 细胞中复制。然而，通过转染来自FCV和CaCV的RNA基因组，子代病毒在任何细胞系中均产生，这表明杯状病毒与细胞的早期相互作用可能是其体外细胞趋向性的主要决定因素。3．病毒结合测定：使用第 1 节中产生的 MAb，开发了针对 CaCV 的流式细胞术病毒结合测定。分析表明CaCV能有效地与许可细胞结合，但不与非许可细胞结合。4．生产并鉴定了一种针对犬源性 MDCK 细胞、可阻断 CaCV 感染的单克隆抗体。 MAb 与 CaCV 允许的细胞结合，但不与 CaCV 不允许的细胞结合，并抑制 CaCV 与允许的细胞结合，表明 MAb 识别 CaCV 受体。通过免疫沉淀，MAb 可与两种分子量分别为 76 和 65 kDa 的蛋白质发生反应。

项目成果

Hashimoto, M., et al.: "Genetic analysis of the RNA polymerase gene of caliciviruses from dogs and cats"Journal of Veterinary Medical Science. 61・6. 603-608 (1999)

Hashimoto, M., et al.：“来自狗和猫的杯状病毒的RNA聚合酶基因的遗传分析”《兽医医学杂志》61·6(1999)。

Matsuura, Y., et al.: "Complete nucleotide sequence genome organization and phylogenic analysis of the canine calicivirus"Virus Genes. (In press). (2002)

Matsuura, Y., et al.：“犬杯状病毒的完整核苷酸序列基因组组织和系统发育分析”病毒基因。

Roerink F,Hashimoto M., Tohya Y, and Mochizuki M.: "Genetic analysis of a canine calicivirus : evidence for a new clade of animal calicivruses"Veterinary Microbiology. 69. 69-72 (1999)

Roerink F、Hashimoto M.、Tohya Y 和 Mochizuki M.：“犬杯状病毒的基因分析：动物杯状病毒新分支的证据”兽医微生物学。

Roerink F, Hashimoto M., Tohya Y, Mochizuki M.: "Organization of the canine calicivirus genome from the RNA polymerase gene to the poly (A) tail."Journal of General Virology. 80. 929-935 (1999)

Roerink F、Hashimoto M.、Tohya Y、Mochizuki M.：“犬杯状病毒基因组从 RNA 聚合酶基因到聚 (A) 尾的组织。”普通病毒学杂志。

Matsuura et al: "Expression and processing of the canine calicivus capsid precursor"J.Gen.Vivol.. 81・1. 195-199 (2000)

Matsuura 等：“犬杯状衣壳前体的表达和加工”J.Gen.Vivol.. 81・199 (2000)。