A Comparative Analysis of Methods for Titering Reovirus

Abstract

Background: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers.

Objective: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT₅₀) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID₅₀) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology.

Methods: Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI₅₀) values of reovirus infected L929 cells with TCID₅₀ titers were analyzed by RTCA, the CI₅₀-TCID₅₀ standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT₅₀-TCID₅₀ standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID₅₀ method.

Results: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL.

Conclusion: The CIT₅₀-TCID₅₀ standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID₅₀ method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID₅₀ method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

Keywords: Cytolytic effect; RTCA technique; TCID50; TRPS; reovirus; virus titer.