Fei tecnai spirit tem t12

The rNDV-IBV-T/B was propagated in nine-day-old SPF chicken embryos and purified by sucrose gradient centrifugation according to a previous report [40 (link)]. Briefly, the harvested allantoic fluid was centrifuged at 1500× g for 20 min to remove the debris and collect the supernatant. Four milliliters of rNDV-IBV-T/B were layered on top of a step gradient of 60%, 50%, 40%, and 25% w/v sucrose prepared in Milli-Q water. Ultracentrifugation was carried out at 120,000 ×g in a SW41 Ti rotor (Beckman Coulter, CA) for 4 h, at 4 °C. The virus-containing band between the 40% and 50% sucrose layers was collected and 5 μl of rNDV-IBV-T/B virus was placed onto the shiny side of an EM grid and then adsorbed for ~3 min. The grid was stained immediately after virus adsorption 3 times with 2% uranyl acetate for 45 s. After negative staining, observation was by an 80 kV FEI Tecnai Spirit TEM T12 (Thermo Fisher) at 98,000× magnification.

To further confirm the morphological characteristics of the thermostable rLS-T-HN-T/B strain, visualization of the thermostable recombinant virus particles and morphological features was performed by TEM detection. The rLS-T-HN-T/B and parental LaSota viruses were purified primarily by sucrose gradient centrifugation according to a previous report [29 (link)]. In brief, viral particles collected from the allantoic fluid were purified by ultracentrifugation in a 40–60% (w/w) sucrose gradient (120,000× g for 2 h). The purified viral particles were pelleted by centrifugation at 100,000× g under 4 °C for 4 h and resuspended in a phosphate-buffered saline (PBS) solution. Ten microliters of the rLS-T-HN-T/B or LaSota strains were placed onto the shiny side of an electron microscopy grid and then adsorbed for ~5 min. The virus adsorbed three times with 2% uranyl acetate for 45 s and stained the grid promptly. The 98,000× magnification images were visualized by the 80 kV FEI Tecnai Spirit TEM T12 (Thermo Fisher, Waltham, MA, USA).