Virus Field Isolates and Designed Host Cells

Sometimes it is necessary not to use laboratory viruses but to follow the evolution of strains within a swarm of viruses from actual patients during therapy or disease progression. Using these viral isolates as "black boxes", without the need for sequencing or detailed characterization, is an advantage of cell-based assays. Enzymatic assays with recombinant proteins or molecular modeling require material and knowledge about each viral strain that significantly complicate the test system, or delay its implementation.

For example, a designed cell line provided important insight into the properties of virus isolates from patients under monotherapy with a reverse transcriptase drug: HeLa was made susceptible to HIV infection by stable expression of CD4 receptor. Monolayers of the CD4-positive HeLa readily form plaques upon HIV infection. Quantification of the plaques with crystal violet-stained cells is a measurement of infectious input, and reduction of plaque formation a measurement of drug efficiency [79]. Although this readout is cumbersome and not amenable to high-throughput systems with current technologies (it may be in the future with improved automated image interpretation), there is a decisive advantage already: most laboratory strains of HIV are selected for fast replication and high cytotoxicity, as these properties facilitate research. Field isolates however - as devastating as they are in human patients - can be relatively slow and innocuous in cell cultures [79, 80]. For such viruses (as opposed to laboratory strains), high-throughput-amenable quantification of the cytopathic effect via reduction of metabolic conversion of dye (e.g., the tetrazolium salt, MTT) by viable cells produces an inadequate signal. However, plaque reduction after the addition of drug allowed the quantification of increased resistance to zidovudine ofHIV in human patients. This assay further allowed the susceptibility ofvarious strains to various reverse-transcriptase inhibitors to be compared. The results suggested that resistance to one compound does not imply resistance to related reverse-transcriptase inhibitors [81]. Narrow opportunities for the development of resistance across different compounds therefore allows combination therapy with several reverse-transcriptase inhibitors (e.g., the nucleoside analogues AZT, 3TC, and abacavir are combined into single formulations).

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