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November 15, 2010 (Vol. 30, No. 20)

New Method for Rapid Virus Quantification

Flow Cytometer Designed to Improve Measurement in Liquid Samples

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    Figure 2. Comparison of log-scale assay results for eight dilutions of influenza A/California/04/2009 (H1N1). Virus Counter results for H1N1 versus TCID50 concentration are shown with red triangles, and H1N1 versus TEM concentration results are shown with blue diamonds. Reported Virus Counter results are mean values (n=3) with the error bars indicating ±1σ from the mean.

    An example log-log plot, showing the relationship of Virus Counter results to TEM and TCID50 results for H1N1 is shown in Figure 2. This data shows that Virus Counter results correlate well (R2=0.98 and slope=0.93) with both expected TEM and TCID50 results over a broad range of virus concentration. These results were also used to back calculate the stock concentration of H1N1 (1.6 ± 0.6) x 1010 vp/mL, which is in agreement with the manufacturer’s reported TEM titer (1.6 x 1010 vp/mL).

    Similar dilution analyses were done for each virus, with all samples yielding linear regression fits to the data with R2≥0.98”, slopes between 0.94 and 1.40, and back calculated Virus Counter results listed in the Table. The R2 values and slopes confirm a strong positive correlation and linear relationship between Virus Counter results and the dilution values calculated from the reported TEM and TCID50 concentrations.

    In general, Virus Counter results are greater than TCID50 and similar to or lower than TEM values. For example, RSV with a genome size of 15 kb ssRNA had a Virus Counter concentration of (4.6 ± 0.6) x 1010 vp/mL, which was equal to the TEM result (4.6 x 1010 vp/mL). CMV, with a genome size of 200 kb dsDNA, measured (3.3 ± 0.5) x 1010 vp/mL on the Virus Counter, which was only slightly lower than the TEM concentration of 4.5 x 1010 vp/mL. These results demonstrate the ability of the Virus Counter to quantify virus samples with a broad range of genome diversity in a fraction of the time required by standard assays.

    It should be noted that while this study was conducted with purified commercially available samples, the Virus Counter has successfully analyzed virus samples from a variety of sources. These real-world samples include viruses in more complex matrices provided by customers and collaborators.

    For example, a baculovirus sample provided by a collaborator in centrifuge-clarified growth media with a reported plaque titer of 1.7 x 108 pfu/mL, measured (5 ± 4) x 109 vp/mL on the Virus Counter and provided a dilution series with excellent correlation and linear relationship (R2=1.0 and slope=1.23) to the plaque titer assay results. 

    In summary, the Virus Counter and associated assay represent a newly available tool for rapid and cost-effective quantification of viruses in liquid samples. The Virus Counter provides a quantitative measure of the number of virus particles per unit volume (vp/mL). The staining process is not virus specific and can be used for the analysis of viruses with highly variable sizes and morphology. Furthermore, the assay, instrument and software are straightforward and do not require a highly trained user.

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