Fig 1.
Analysis of optimal culture conditions.
Note: BHK-21 cells cultured in 96-well plates at a density of 3.3 x 104 cell/cm2 for 24 h were inoculated with 10-times serially diluted WRV (range 0.01–1000 TCDI50/well; 4 wells each dose) in culture medium. Cells were incubated (A) for 72 h in DMEM-HAM supplemented with 2.5% fetal bovine serum (FBS) at 35°C or 37°C, (B) for 72 h in DMEM-HAM added 2.5% or 3% FBS at 37°C, or (C) for 48, 72 ou 96 h in DMEM-HAM 2.5% FBS at 37°C. After incubation, cells were fixed and DFA was carried out to identify viral positive cells. TCID50, tissue culture infective dose 50%.
Table 1.
Effects of subcultures on residual live virus recovery.
Fig 2.
Determination of limit of detection of in vitro assay.
Note 1: Percentage of positive wells from DFA assay (gray bars) and quantification rates (log10) of RABV at 72 h of incubation with duplex RT-qPCR (black line) calculated as the mean from 3 independent experiments. Note 2. For the sample to be considered positive in the DFA test, it must have at least one positive well, while for RT-qPCR positivity, the quantification obtained in RT-qPCR at 72 h p.i. must be above the quantitation limit and higher than that observed at 0 h”.
Fig 3.
Comparison of sensitivity between in vivo and in vitro assays.
Note: Percentage of positive mice in the in vivo assay was calculated as (number of dead mice/56 adult and neonatal mice) x 100 and that of well in the in vitro assay was calculated as (number of positive wells/92) x 100. Data is presented as the mean of 3 independent experiments.
Fig 4.
Evaluation of inactivated viral suspension samples.
Note: Percentage of positive wells in in vitro viral recovery assay (black squares) and quantification rates (log10) of RABV by RT-qPCR at 0 h (dark gray bars) and at 72 h (light gray bars) of incubation. PC, positive control– 2 μL of WRV, 105,78 TCID50/mL; NC, negative control–culture medium.
Fig 5.
Note: cells incubated with (Group A) culture medium–negative control, (Group B) 2 μL of WRV, 105,78 TCID50/mL–positive control, (Group C) satisfactory IVS sample, and (Group D) unsatisfactory IVS sample, 72 h p.i. Original magnification, 40x.
Fig 6.
Transmission Electronic Microscopy of BHK-21 cells.
Note: cells incubated with (A) DMEM-HAM 2.5% FBS–negative control (1 μm), (B1 and 2) WRV–positive control (1 μm and 200 nm), (C) IVS–(1 μm) and (D1 and 2) IVS+ (1 μm and 200 nm) at 37°C for 72 h. Arrowheads in (B) indicate rabies virus particle, arrowheads in (D) indicate unconfirmed virus particles.
Fig 7.
Confirmation of infectivity in vivo.
Note: (A) mice injected intra-cerebrally with IVS–, 21 days after inoculation showing no clinical signs of rabies; (B) mice injected intra-cerebrally with IVS+, 5 days after inoculation showing specific rabies symptoms, such as ruffled fur, hunched back, slow movements and paralysis; (C) brain DFA of mouse injected intra-cerebrally with IVS–(40x); (D) brain DFA of mouse injected intra-cerebrally with IVS+ (40x); (E) brain TEM of mouse inoculated with IVS–(1 μm); (F) brain TEM of mouse inoculated with IVS+, arrow indicates rabies virus particles (200 nm); (G) brain H&E stain of mouse inoculated with IVS–, basal ganglia with normal cellularity (50x); (H) brain H&E stain of mouse inoculated with IVS+, Purkinje cell with Negri body–arrow (400x).