Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA
Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA
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DOI：
10.1128/jvi.78.15.8264-8270.2004
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发表时间：
2004-08-01
影响因子：
5.4
通讯作者：
Fouchier, RAM
中科院分区：
文献类型：
--
作者：
Herfst, S;de Graaf, M;Fouchier, RAM
人偏肺病毒（hMPV）是一种新发现的与呼吸道疾病相关的病原体，主要发生在幼儿、免疫功能低下者和老年人中。没有末端前导序列和尾部序列的原型hMPV分离株NL/1/00的基因组序列先前已报道。在这里，我们描述了两个hMPV分离株，NL/1/00和NL/1/99，代表hMPV的两个主要遗传谱系的前导和尾随序列。产生了微型基因组构建体，其中绿色荧光蛋白或氯霉素乙酰转移酶基因的侧翼是来源于两种hMPV谱系的病毒基因组末端，并使用T7 RNA聚合酶启动子-终止子盒转录。在293 T或幼仓鼠肾细胞中，将微型基因组构建体与表达聚合酶复合物组分L、P、N和M2.1的质粒共转染导致报告基因的表达。当用NL/1/00或NL/1/99病毒基因组的有义或反义全长cDNA拷贝替换微型基因组时，从转染的细胞中回收重组病毒。病毒滴度高达10（7.2）和10（5.7）50%组织培养感染剂量/ml，分别与正义和反义质粒。重组病毒在Vero细胞中复制的动力学与亲本病毒相似。这种反向遗传学系统为应用和基础研究提供了一种重要的新工具。
Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.