Brown David D, Rima Bertus K, Allen Ingrid V, Baron Michael D, Banyard Ashley C, Barrett Thomas, Duprex W Paul
School of Biomedical Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom.
J Virol. 2005 Nov;79(22):14330-8. doi: 10.1128/JVI.79.22.14330-14338.2005.
Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.
负链RNA病毒编码一种单一的RNA依赖性RNA聚合酶（RdRp），该聚合酶负责转录和复制基因组。将来自恶性牛瘟病毒（RPV）野生型毒株的编码RdRp的开放阅读框插入到一个表达质粒中。将编码增强型绿色荧光蛋白（EGFP）的序列插入到RdRp的一个可变铰链区。所得的聚合酶具有自发荧光，并且其在合成微型基因组复制/转录中的活性降低。我们研究了通过将编码修饰后的RdRp的DNA序列插入到一个全长反基因组质粒中来合理减毒病毒的潜力，从该质粒中可以拯救出一种恶性病毒（rRPV(KO)）。拯救出了一种重组病毒rRPV(KO)L-RRegfpR，其在体外的生长速度和滴度与rRPV(KO)无明显差异。在大的细胞质内含物和细胞膜下方可见荧光标记的聚合酶。将10(4) TCID(50)的rRPV(KO)亲本重组病毒皮下注射到牛体内，会导致严重的疾病症状（白细胞减少/腹泻和发热），并在感染后9天死亡。感染rRPV(KO)L-RRegfpR的动物表现出短暂的白细胞减少和轻度发热，唯一明显的临床症状是一只眼睛中度发红和轻微的眼鼻分泌物。从外周血淋巴细胞和眼拭子中分离出了表达修饰后聚合酶的病毒。这表明一种恶性麻疹病毒仅通过调节RdRp活性就可以在一步中减毒，并且体外病毒生长与体内病毒生长之间不一定存在相关性。
