Novel, real-time cell analysis for measuring viral cytopathogenesis and the efficacy of neutralizing antibodies to the 2009 influenza A (H1N1) virus - PubMed (original) (raw)

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Novel, real-time cell analysis for measuring viral cytopathogenesis and the efficacy of neutralizing antibodies to the 2009 influenza A (H1N1) virus

Di Tian et al. PLoS One. 2012.

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Abstract

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×10(2) to 5.50×10(7) copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

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

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.

Figure 3

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

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.

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