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.
Hybrid Seven-bar Press Mechanism