Rapid baculovirus titration assay based on viable cell side scatter (SSC)

Qi, Jing; Liu, Tao; Pan, Junjie; Zhang, Chun

doi:10.1016/j.aca.2015.04.007

Cited by 8 publications

(7 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…transport of intracellular nucleic acids, proteins, and other biological resources. Here, we used SSC to characterize cells and found that cells with high SSC contained more viral proteins and viral RNA copies and a higher ratio of infectivity-positive cells than cells with medium or low SSC, and this result is consistent with a previous study showing that viral titers increased with increased SSC in baculovirus-infected insect cells (33). Correlations between virus and host cell cycle have also been studied.…”

Section: Discussionsupporting

confidence: 90%

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus

Xin

Wang

Han

et al. 2018

J Virol

View full text Add to dashboard Cite

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G/M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells.

show abstract

Section: Discussionsupporting

confidence: 90%

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus

Xin

Wang

Han

et al. 2018

J Virol

View full text Add to dashboard Cite

show abstract

“…Existing baculovirus titration methods have significant drawbacks. Traditional plaque and endpoint dilution assays require 5-10 days for results, and alternatives that are faster count both infectious and non-infectious particles (Shen et al, 2002), or require maintenance of an additional cell line (Hopkins and Esposito, 2009), specialized equipment (Qi et al, 2015) or relatively expensive antibody-based reagents (Wang et al, 2013). In any major structural biology undertaking, many expression conditions and constructs need to be assayed; thus, any way to increase efficiency can dramatically increase the likelihood of success.…”

Section: Resultsmentioning

confidence: 99%

Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins

2016

View full text Add to dashboard Cite

The ability of oligomeric membrane proteins to assemble in different functional ratios of subunits is a common feature across many systems. Recombinant expression of hetero-oligomeric proteins with defined stoichiometries facilitates detailed structural and functional analyses, but remains a major challenge. Here we present two methods for overcoming this challenge: one for rapid virus titration and another for stoichiometry determination. When these methods are coupled, they allow for efficient dissection of the heteromer stoichiometry problem and optimization of homogeneous protein expression. We demonstrate the utility of the methods in a system that to date has proved resistant to atomic-scale structural study, the nicotinic acetylcholine receptor. Leveraging these two methods, we have successfully expressed, purified, and grown diffraction-quality crystals of this challenging target.

show abstract

“…The titer of baculoviral stock is an essential parameter during transduction because the transduction dosage in MOI is obtained from the titer, and therefore it is necessary to measure the titer of any batch of viral stock. In previous studies, various methods have been applied to quantitate recombinant baculoviruses, such as the traditional plaque assay [18], flow cytometry assay [19], immunological assay [20], MTT assay [21], alamar blue assay [21], quantitative real-time PCR [21,22], methods based on visible cell sizes [21,23], viable cell side scatter [24], etc., most of which take up to several days, require expensive detection kits, or entail multiple laborious operation steps. In this study, the titer of baculoviral stock was quantitated based on an end-point dilution method with fluorescence, which is sensitive and rapid because of the robust promotor and luminous fluorescent signals, economical due to the simple reagent, and convenient to operate, as previous studies have demonstrated [21,25,26].…”

Section: Discussionmentioning

confidence: 99%

Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

Wang

Fang

Pan

et al. 2018

IJMS

View full text Add to dashboard Cite

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

show abstract

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

Cited by 8 publications

References 21 publications

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus

Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins

Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

Contact Info

Product

Resources

About