Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture.
Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture.
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DOI：
10.1099/vir.0.018994-0
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发表时间：
2010-06
期刊：
影响因子：
--
通讯作者：
Davison AJ
中科院分区：
文献类型：
--
作者：
Dargan DJ;Douglas E;Cunningham C;Jamieson F;Stanton RJ;Baluchova K;McSharry BP;Tomasec P;Emery VC;Percivalle E;Sarasini A;Gerna G;Wilkinson GW;Davison AJ
通过从临床样品中分离四种毒株，在各种细胞类型中传代，并对最终传代的十个完整病毒基因组进行测序，监测人巨细胞病毒适应细胞培养期间发生的突变。通过中间传代和原始临床样本的靶向测序评估突变动力学。基因RL13和UL128基因座（UL128L，由基因UL128、UL130和UL131A组成）在所有菌株中突变。RL13中的突变发生在成纤维细胞、上皮细胞和内皮细胞中，而UL128L中的突变仅限于成纤维细胞，并且比RL13中的突变晚被检测到。另外，在三个菌株中，含有基因UL145、UL144、UL142、UL141和UL140的区域发生突变。所有菌株在基因组的其他区域都表现出许多突变，在部分反向重复序列中占优势。对源自所选传代的病毒的动力学生长产率进行了研究，所述传代主要在RL13和UL128L中未突变（RL13+UL128L+），或在RL13中大量突变（RL13−UL128L+），或在RL13和UL128L中均突变（RL13−UL128L−）。RL13−UL128L−病毒比RL13−UL128L+病毒产生更高的感染性子代产量，RL13−UL128L+病毒比RL13+UL128L+病毒产生更高的产量。这些结果强烈地表明，RL13和UL128L对成纤维细胞的生长发挥至少部分独立的抑制作用。由于所有分离株在所有检测的细胞类型中均证明遗传不稳定，因此建议在选择和监测用于脆弱功能实验研究的菌株时应谨慎，特别是涉及细胞嗜性、免疫逃避或生长节制的菌株。
Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13−UL128L+) or both RL13 and UL128L (RL13−UL128L−). RL13−UL128L− viruses produced greater yields of infectious progeny than RL13−UL128L+ viruses, and RL13−UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.
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