期刊
BIOTECHNIQUES
卷 75, 期 5, 页码 183-194出版社
FUTURE SCI LTD
DOI: 10.2144/btn-2023-0046
关键词
cell-free infectivity; clinical isolates; cytomegalovirus; shRNA; UL128; virions
The authors demonstrate that transduction of fibroblasts with UL128-specific-shRNA allows the release of cell-free infectivity of recent isolates of human cytomegalovirus (HCMV), enabling applications previously unsuitable for these isolates.
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 UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-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 UL128-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, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.
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