Analysis of factors determining the host range of calicviruses

这项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寻找决定杯状病毒宿主特异性的病毒和细胞因子。在本研究中，我们获得了以下几个新的发现。犬杯状病毒的分子生物学分析：测定犬杯状病毒基因组全序列。用单抗鉴定的CACV的两个构象中和表位位于猫杯状病毒衣壳蛋白的5‘和3’高变区。FCV和CACV的体外宿主范围：CACV只能在犬器官细胞系中复制。FCV可以在猫细胞系中很好地生长，一些FCV菌株可以在猿源的Vero细胞中复制。然而，通过转染FCV和CACV的RNA基因组，在任何细胞系中都能同样产生后代病毒，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嗜性的主要决定因素。病毒结合试验：利用第一节中制备的单抗，建立了CACV的流式细胞术病毒结合试验。分析表明，CACV能有效地与允许细胞结合，但不能与非允许细胞结合。制备并鉴定了一种能阻断CACV感染的犬源性MDCK细胞的单抗。该单抗可与CACV的许可细胞结合，但不与CACV的非许可细胞结合，并抑制CACV与许可细胞的结合，表明MAb识别CACV受体。免疫沉淀法表明，该单抗可与两种相对分子质量分别为76和65 kDa的蛋白质发生反应。

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.