Journal: Vaccines

Article Title: Avian Cell Line DuckCelt ® -T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac ®

doi: 10.3390/vaccines9101190

Figure Lengend Snippet: Viral kinetics of wild-type C-85473 HMPV and recombinant rC-85473-GFP HPMV in the DuckCelt ® -T17 cell line. Cells seeded at 1 × 10 6 cell/mL in 10 mL working volume and infected at an MOI of 0.01. Trypsin added at 0.5 µg/mL on Day (D) 0, D2, D4, and D7 (+). ( a ) Picture of T17 cells infected with rC-85473-GFP at 7 days post-infection (dpi). Each picture was taken in bright field and fluorescent microscopy (20× magnification, scale bars represent 150 µm); ( b ) viral titers measured from culture medium as 50% tissue culture infectious doses (TCID50)/mL in LLC-MK2 cells; ( c ) cell growth (solid line) and viability percentage (dotted line) measured with the Countess™ II FL Automated Cell Counter. Results shown as means ± SD and represent duplicates in two independent experiments. *** p < 0.001 when comparing the infected conditions to ( b ) each other or ( c ) the mock condition using two-way repeated measures ANOVA.

Article Snippet: Twenty mice were infected by intranasal instillation with 1 × 10 6 TCID 50 of Metavac ® viruses produced in LLC-MK2 adherent cells (Metavac ® LLC) or in in-suspension DuckCelt ® -T17 cells (Metavac ® T17).

Techniques: Recombinant, Infection, Microscopy

Journal: Vaccines

Article Title: Avian Cell Line DuckCelt ® -T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac ®

doi: 10.3390/vaccines9101190

Figure Lengend Snippet: Evaluation of multiplicity of infection (MOI), cell density, trypsin concentration, and optimal timing for trypsin addition on rC-85473-GFP HMPV production kinetics in the DuckCelt ® -T17 cell line. Culture parameters in a 10 mL working volume were separately evaluated by ( a , c , e , g ) viral titration, and (( b , d , f , h ) cell viability and infectivity measurement; dotted line for viability, and solid line for infectivity). Viral titers measured from culture medium as TCID50/mL in LLC-MK2 cells. Viability measured with trypan blue using an automated cell counter. Infected GFP-positive cells evaluated with the FACS CantoII. ( a , b ) Evaluation of MOI of 0.1, 0.01, or 0.001. ( c , d ) Evaluation of three different cell densities at time of infection: 0.5, 1, or 4 × 10 6 cell/mL. (e–f) Evaluation of timing of trypsin addition. Trypsin added at 0.5 µg/mL at D0, at D0 and D4, or at D0, D4, and D7, or no trypsin was added. ( g , h ) Evaluation of trypsin concentration: 0.5, 2, or 4 µg/mL. Results shown as means ± SD and represent duplicates in two independent experiments. * p < 0.05, *** p < 0.001 when comparing infected conditions to each other using a two-way repeated-measures ANOVA.

Article Snippet: Twenty mice were infected by intranasal instillation with 1 × 10 6 TCID 50 of Metavac ® viruses produced in LLC-MK2 adherent cells (Metavac ® LLC) or in in-suspension DuckCelt ® -T17 cells (Metavac ® T17).

Techniques: Infection, Concentration Assay, Titration

Journal: Vaccines

Article Title: Avian Cell Line DuckCelt ® -T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac ®

doi: 10.3390/vaccines9101190

Figure Lengend Snippet: Visualization of T17-produced rC-85473-GFP and Metavac ® virus particles, and evaluation of in vitro replicative capacity. ( a ) Representative negative stain electron microscopy images of rC-85473-GFP and Metavac ® virions, obtained from DuckCelt ® -T17 culture. Bar represents 100 nm. Enlargements of viral membrane shown as inset, and glycoproteins indicated by white arrowheads; ( b , d ) LLC-MK2 monolayers in 24-well plates infected with each of the recombinant HMPVs at an MOI of 0.01. ( b ) Images of representative cytopathic effects of each virus captured after 4 dpi by fluorescent microscopy (10× magnification, scale bars represent 250 µm). ( c ) Supernatants harvested every 24 h for 7 days, frozen, and subsequently thawed and titrated as TCID 50 /mL onto LLC-MK2 cells. Growth curves represent mean titers ± SD of 3 biological replicates, titrated at each time point. ( d ) Infected LLC-MK2 monolayers fixed in formaldehyde after harvest, and images captured after crystal violet coloration.

Article Snippet: Twenty mice were infected by intranasal instillation with 1 × 10 6 TCID 50 of Metavac ® viruses produced in LLC-MK2 adherent cells (Metavac ® LLC) or in in-suspension DuckCelt ® -T17 cells (Metavac ® T17).

Techniques: Produced, In Vitro, Staining, Electron Microscopy, Infection, Recombinant, Microscopy