strains ra2  (ATCC)

Journal: Viruses

Article Title: The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

doi: 10.3390/v10080446

Figure Lengend Snippet: Assessment of sample preparation conditions for cryo-EM data collection. ( A ) One-step growth curve of RSV A2 in BEAS-2B cells over a period of 36 h at a M.O.I. of 10. Infected cells were harvested at indicated time points and titrated in Vero cells. ( B – D ) Flow cytometry, viral titer, and RNA quantification analysis of RSV-infected BEAS-2B cells, at indicated M.O.I.s after 24 h.p.i. These data indicate that at a M.O.I. of 10, infectious particles are produced at 24 h.p.i., suggesting that the particles being imaged by cryo-EM are infectious. ( B ) Relative ratio of cells infected at indicated M.O.I. by flow cytometry using mK + signal, represented by the ratio of infected cells over the total cells. ( C ) Viral titer at 24 h.p.i. determined by FFU using mK + signal. ( D ) Relative amount of viral RNA from infected cells determined by real time RT-PCR represented by inverse C T values (1/C T ). C T value is the number of cycles needed to pass the background level. For all 4 experiments, error bars represent the standard deviation of 3 independent experiments.

Article Snippet: For cryo-EM sample optimization studies, BEAS-2B cells were seeded at 1.6 × 10 5 cells/well in 6-well plates, infected with the rA2-mK + strain at various multiplicities of infection (M.O.I.), and incubated for 24 h at 37 °C and 5% CO 2 .

Techniques: Sample Prep, Cryo-EM Sample Prep, Infection, Flow Cytometry, Produced, Quantitative RT-PCR, Standard Deviation

Journal: Viruses

Article Title: The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

doi: 10.3390/v10080446

Figure Lengend Snippet: Representative montages showing 2D projections of RSV-infected cells. ( A ) Representative montages showing released viruses from RSV-infected cells. Montage view of released viral particles captured from RSV A2 infected A549 cells. Sample was prepared as described in the method section. RSV morphology varies, with the overall structure being filamentous. Filaments may be straight, bent, or branched. ( B ) Montage view of the assembly site from RSV-infected cells with the F glycoprotein immunogold labeled. The region imaged is an active assembly site, with RSV filaments extending from the cell plasma membrane (dashed yellow line). ( C ) Zoomed-in view of the boxed region in B. Note the presence of 6-nm immunogold on the RSV filaments (white arrows), and the absence of gold particles on cell extension (black arrows). Scale bars are 2 µm ( A ), 1 µm ( B ), and 500 nm ( C ).

Article Snippet: For cryo-EM sample optimization studies, BEAS-2B cells were seeded at 1.6 × 10 5 cells/well in 6-well plates, infected with the rA2-mK + strain at various multiplicities of infection (M.O.I.), and incubated for 24 h at 37 °C and 5% CO 2 .

Techniques: Infection, Labeling

Journal: Viruses

Article Title: The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

doi: 10.3390/v10080446

Figure Lengend Snippet: Cryo-ET of released RSV filaments from RSV-infected cells. ( A – F ) Cell lines including HeLa ( A , F ) and lung derived cell lines A549 ( B ), MRC-5 ( C ), and BEAS-2B ( D , E ), were infected with RSV A2 strain ( A – D ) or RSV TN strain ( E , F ). Among these samples, filamentous particles were consistently observed under frozen-hydrated conditions. Scale bars are 200 nm.

Article Snippet: For cryo-EM sample optimization studies, BEAS-2B cells were seeded at 1.6 × 10 5 cells/well in 6-well plates, infected with the rA2-mK + strain at various multiplicities of infection (M.O.I.), and incubated for 24 h at 37 °C and 5% CO 2 .

Techniques: Tomography, Infection, Derivative Assay