Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus
Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus
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DOI：
10.1128/jvi.72.1.380-387.1998
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发表时间：
1998-01-01
影响因子：
5.4
通讯作者：
Moormann, RJM
中科院分区：
文献类型：
--
作者：
Meulenberg, JJM;BosDeRuijter, JNA;Moormann, RJM
测定了猪繁殖与呼吸综合征病毒莱利斯塔病毒分离株（LV）基因组RNA的5 '末端。为了构建全长cDNA克隆，将5 '末端序列连接到覆盖LV全基因组的cDNA克隆。当从这些全长cDNA克隆体外转录的RNA转染到BHK-21细胞中时，产生并分泌感染性LV。通过传代至猪肺泡肺巨噬细胞或CL 2621细胞来拯救病毒。当感染性转录本转染到猪肺泡肺巨噬细胞或CL 2621细胞时，由于这些细胞的转染效率差，没有产生感染性病毒。用LV cDNA的感染性转录本转染的BHK-21细胞产生的病毒的生长特性类似于cDNA来源的亲本病毒的生长特性。两个核苷酸的变化，导致一个独特的PacI限制性位点直接下游的ORF 7基因被引入基因组长度的cDNA克隆。从该突变的cDNA克隆中回收的病毒保留了PacI位点，这证实了从克隆的cDNA重新产生感染性LV。这些结果表明，LV的感染性克隆使我们能够在特定位点诱变病毒基因组，因此，它将是有用的病毒的详细分子特征，以及用于猪的安全和有效的活疫苗的开发。
The 5'-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5'-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.