CIRAD, UMR ASTRE, F-34398 Montpellier, France; INRA, UMR1309 ASTRE, F-34398 Montpellier, France.
CIRAD, UMR ASTRE, F-97170 Petit-Bourg, Guadeloupe, France; INRA, UMR1309 ASTRE, F-34398 Montpellier, France.
J Virol Methods. 2017 Nov;249:111-116. doi: 10.1016/j.jviromet.2017.08.024. Epub 2017 Sep 1.
Rescue of negative-sense single-stranded RNA viruses ((-)ssRNA virus), generally requires the handling of a large number of plasmids to provide the virus genome and essential components for gene expression and genome replication. This constraint probably renders reverse genetics of (-)ssRNA virus more complex and less efficient. Some authors have shown that the fewer the plasmids, the more efficient reverse genetics is for segmented RNA virus. However, it is not clear if the same applies for (-)ssRNA, such as Newcastle disease virus (NDV). To address this issue, six variants of NDV reverse genetic systems were established by cloning combinations of NP, P and L genes, mini-genome or full-genome in 4, 3, 2 and 1 plasmid. In terms of mini-genome and full-genome rescue, we showed that only the 2-plasmid system, assembling three support plasmids together, was able to improve the rescue efficiency over that of the conventional 4-plasmid system. These results may help establish and/or improve reverse genetics for other mononegaviruses.
负义单链 RNA 病毒 ((-)ssRNA 病毒)的拯救通常需要处理大量的质粒，以提供病毒基因组和用于基因表达和基因组复制的必需成分。这种限制可能使 (-)ssRNA 病毒的反向遗传学更加复杂和低效。一些作者已经表明，质粒越少，分段 RNA 病毒的反向遗传学效率就越高。然而，对于 (-)ssRNA 病毒，如新城疫病毒 (NDV)，情况是否相同尚不清楚。为了解决这个问题，通过将 NP、P 和 L 基因、迷你基因组或全长基因组克隆组合在 4、3、2 和 1 个质粒中，建立了六种 NDV 反向遗传系统的变体。在迷你基因组和全长基因组拯救方面，我们表明，只有组装三个支持质粒的 2 质粒系统能够提高拯救效率，优于传统的 4 质粒系统。这些结果可能有助于建立和/或改进其他单负链 RNA 病毒的反向遗传学。
