Institute of Virology, Ulm University Medical Center, Ulm, Germany.
Biotechniques. 2023 Nov;75(5):183-194. doi: 10.2144/btn-2023-0046. Epub 2023 Oct 17.
Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral gene locus. The authors transduced fibroblasts with a lentiviral vector encoding -specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with -shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, -shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.
与最近分离的人巨细胞病毒（HCMV）合作，由于其严格的细胞相关生长，且上清液中缺乏感染力，因此变得复杂。适应无细胞生长与病毒基因座的破坏有关。作者通过慢病毒载体转导成纤维细胞，该载体编码特异性 shRNA，可在不改变遗传物质的情况下释放无细胞感染力。转导细胞与含有细胞相关分离株的成纤维细胞共培养，并通过免疫印迹验证 UL128 蛋白的敲低。与对照相比，与 shRNA 共培养的分离株中细胞游离感染性增加了 1000 倍，并且可以通过密度梯度纯化病毒粒子。转导的成纤维细胞还可以直接从临床标本中分离 HCMV，并将其无细胞转移到其他细胞类型。总之，shRNA 转导的成纤维细胞允许以前不适合最近分离株的应用。
