Figure 1.
Dynamic monitoring of MDCK cell proliferation.
MDCK cells were seeded in the E-Plates and continuously monitored by measuring the CI to identify the appropriate cell numbers, TPCK-trypsin concentration, and the time point for addition of virus (i.e., during growth or early stationary phase). A) Proliferation curves of MDCK cells in cell culture media. B) Proliferation curves of MDCK cells in SFM with TPCK-trypsin. At the indicated time point the cells were inoculated with different concentrations of virus and TPCK-trypsin.
Figure 2.
Real-time monitoring of pH1N1 virus-mediated cytopathogenicity.
A) The CPE on MDCK cells infected with SH37T (1∶10 diluted) at 24, 48, and 72 hours post-infection. B) Real-time monitoring of the CPE on MDCK cells infected with SH37T using the RTCA system. The curve was an average of four independent replicate wells. At the indicated time point the cells were inoculated with different dilutions of virus. C) The colorimetric assays for the CPE on MDCK cells infected with SH37T (1∶10 diluted). OD values at A450 obtained at 24, 48, and 72 hours post-infection are shown. Error bars indicate standard deviation. D) Difference in CPE kinetic patterns between SH37T and SH143T with the same infectious vial load of 5.50×107 copies/mL. The plotted lines indicate the different time points when the CI declined to 0 between stains.
Table 1.
TCID50, viral load, and the ratio viral load per TCID50 of two H1N1 stains.
Figure 3.
Typical results of real-time neutralization tests against SH37T.
CPE kinetic patterns of MDCK cells inoculated with 100 TCID50 virus incubated with a series dilution of sera samples. Antibody titers were calculated as a function of CI with a sequential dilution series of S0 (A), S1 (B), and S2 (C). The curve was an average of three independent replicate wells. At the indicated time point the cells were inoculated with the serial serum-virus mixture. Three replicate wells of 1280-fold diluted S2 treated cells showed different curves (D). Cell control: MDCK cells only; virus control: absence of test sera.
Figure 4.
Antibody titers in 21 pre- and post-vaccination sera triples measured by real-time NT and HI tests.
A, B) There was a statistically significant difference (p<0.01, Wilcoxon signed rank test) when the pre- and post-vaccination titers obtained by real-time NT (A) or HI test (B) were compared to each other. C) The NT and HI antibody titers correlated well (Spearman's correlation r = 0.78, p<0.01). D) Antibody titers on day 21 after vaccination relative to baseline (day 0). There was no significant difference (p>0.05, Wilcoxon signed rank test) in the results among the NT versus HI tests. Low responders: no increase above twofold; high responders: fourfold or more above baseline.
Table 2.
Geometric mean titers (GMT), titer ranges, and significant titer rises observed from 21 pre- and post-vaccination serum triples.