Purification of human respiratory syncytial virus: superiority of sucrose gradient over percoll, renografin, and metrizamide gradients
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Steady-state persistence of respiratory syncytial virus in a macrophage-like cell line and sequence analysis of the persistent viral genome
2021, Virus ResearchCitation Excerpt :Additional monitoring of hRSV proteins in sucrose gradients was performed by Western blot, detecting F, N and M, in fractions 35–43 (in some cases F protein was not detected in fractions 35 and 36), whereas fractions 1–34 were negative (Fig. 4B). Reported buoyant density for hRSV is 1.17–1.20 g/mL (Mbiguino and Menezes, 1991) and we calculated average density of 1.068 ± 0.007 g/mL (range 1.045–1.100 g/mL) for viruses in MΦP cultures (Fig. 4A). As a control, we also determined buoyant density for hRSV produced in acutely infected HEp-2 cell-cultures (72 h post infection), with viral titer of 2.40 ± 0.55 × 106 TCID50/mL; average buoyant density was 1.202 ± 0.022 g/mL, in accordance with sucrose fractions 6–20, whereas A260 was higher than A280 (Fig. 4C).
Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode
2016, VaccineCitation Excerpt :Thereby, large molecules can be purified from smaller impurities in the negative purification mode. Purification of hRSV has traditionally been performed by ultracentrifugation in either sucrose [16–19] or iodixanol [20], with recoveries of up to 60–70% infectious virus. Chromatographic purification of viral proteins from cell culture-derived RSV has been described [21,22].
Protein impurities from cell culture dramatically impact transduction efficiency of polymer/virus hybrid vectors
2013, Journal of Virological MethodsCitation Excerpt :The aim of the current work was to separate MLV-VLPs from proteins and to study the effect of purification on polymer/MLV-VLP complex formation and transduction. In earlier studies, virus purification was accomplished using density gradient methods such as cesium chloride and sucrose cushion ultracentrifugation (Jeong et al., 2006; Mbiguino and Menezes, 1991). Although these methods are able to separate viral particles from closely related strains, there are disadvantages associated with these methods such as time requirements, low yield, and disruption of functionality of virus particles due to the viscous and hyperosmotic nature of the density gradient agents together with high shear stress (Pedro et al., 2008).
Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications
2014, Microscopy and MicroanalysisGeneration of quality-controlled SARS-CoV-2 variant stocks
2023, Nature Protocols