Evans S A, Stirrups K, Dalton K, Shaw K, Cavanagh D, Britton P
Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, United Kingdom.
Adv Exp Med Biol. 1998;440:687-92. doi: 10.1007/978-1-4615-5331-1_88.
Based on the natural ability of coronaviruses to undergo homologous RNA recombination, we are working to produce infectious bronchitis virus (IBV) recombinants using RNA generated from recombinant fowlpox viruses (FPV). The aim is to replace the spike (S) gene of an existing IBV vaccine strain with the S gene of a heterologous strain. CD-61 is an IBV defective RNA (D-RNA) derived from a naturally occurring IBV D-RNA (CD-91). CD-61 D-RNA is being investigated as an RNA vector for the expression of heterologous genes. T7-derived RNA transcripts of CD-61 can be replicated and passaged in the presence of helper virus, following electroporation into IBV-infected cells. CD-61 cDNA was modified by the addition of the hepatitis delta virus ribozyme plus T7 terminator downstream of the 3'UTR. This allowed the synthesis of discreet RNA transcripts. The complete cassette was cloned into an FPV transfer vector (pEFL10) for generating recombinant fowlpox viruses. FPV/CD-61 recombinants will be assessed for D-RNA production in IBV-infected cells. The luciferase reporter gene sequence has been inserted into the modified CD-61, under the control of the IBV transcription associated sequence (TAS) from gene 5. Luciferase has been successfully expressed from CD-61 in helper virus-infected cells.
基于冠状病毒进行同源RNA重组的天然能力，我们正在致力于利用重组禽痘病毒（FPV）产生的RNA制备传染性支气管炎病毒（IBV）重组体。目的是用异源毒株的刺突（S）基因替换现有IBV疫苗株的S基因。CD - 61是一种源自天然存在的IBV缺陷RNA（D - RNA，CD - 91）的IBV缺陷RNA。CD - 61 D - RNA正在作为一种用于表达异源基因的RNA载体进行研究。在电穿孔导入受IBV感染的细胞后，CD - 61的T7衍生RNA转录本能够在辅助病毒存在的情况下进行复制和传代。通过在3'UTR下游添加丁型肝炎病毒核酶和T7终止子对CD - 61 cDNA进行修饰。这使得能够合成离散的RNA转录本。将完整的盒式结构克隆到一个FPV转移载体（pEFL10）中以产生重组禽痘病毒。将评估FPV/CD - 61重组体在受IBV感染的细胞中产生D - RNA的情况。荧光素酶报告基因序列已在来自基因5的IBV转录相关序列（TAS）的控制下插入到修饰后的CD - 61中。荧光素酶已在辅助病毒感染的细胞中从CD - 61成功表达。
