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Updated: Aug 11, 2026
Optimization of a Quantitative Micro-neutralization Assay
Published on: December 14, 2016
Analysis of the beta-propiolactone sensitivity and optimization of inactivation methods for human influenza H3N2
Yutaka Sasaki1, Naoto Yoshino1, Shigehiro Sato1
1Division of Infectious Diseases and Immunology, Department of Microbiology, School of Medicine, Iwate Medical University, Japan.
Abstract:
Beta-propiolactone (BPL) is used as an inactivating reagent for influenza virus in a number of countries. However, the treatment of viruses with BPL occasionally results in a decrease in the hemagglutinin (HA) titer, which complicates vaccine development. In the present study, we examined the biological and biochemical characteristics of human H1N1 and H3N2 viruses treated with BPL, and developed an inactivation method for BPL-sensitive viruses. A significant decrease in HA titer was detected in the H3N2 viruses examined. The decrease in the pH of the virus fluid was not associated with the decreased HA titer, indicating that the decrease in HA titer for the H3N2 virus is the result of the direct effect of BPL. Excessive modification of M1 by BPL and loss of virion diameter were observed in 0.1% BPL-treated H3N2 virus. Taken together, these results suggest that the BPL sensitivity of H3N2 virus results from disruption of the virion. By contrast, the H3N2 virus was successfully inactivated by 0.02% BPL without a significant decrease in the HA titer or disruption of virion structure. Furthermore, we found that the 0.02% BPL in the virion preparation was hydrolyzed successfully by incubation at 37°C for 7h. Thus, mild treatment with a low concentration of BPL enabled us to inactivate the H3N2 virus.
Insights
Beta-propiolactone (BPL) can inactivate influenza viruses but may reduce hemagglutinin (HA) titers. A mild BPL treatment method was developed to effectively inactivate H3N2 virus without compromising HA levels or virion integrity.
Area of Science:
- Virology
- Vaccine Development
- Biochemistry
Background:
- Beta-propiolactone (BPL) is a common reagent for inactivating influenza viruses.
- BPL treatment can sometimes decrease hemagglutinin (HA) titers, hindering vaccine production.
- Influenza H1N1 and H3N2 viruses exhibit varying sensitivities to BPL inactivation.
Purpose of the Study:
- To investigate the biological and biochemical effects of BPL on human H1N1 and H3N2 influenza viruses.
- To develop an optimized BPL inactivation method for BPL-sensitive influenza strains.
- To understand the mechanisms behind HA titer reduction during BPL treatment.
Main Methods:
- Treatment of H3N2 and H1N1 viruses with varying concentrations of BPL.
- Assessment of HA titers post-BPL treatment.
- Analysis of virion structure and M1 protein modification using electron microscopy and biochemical assays.
- Monitoring of BPL hydrolysis at 37°C for 7 hours.
Main Results:
- H3N2 viruses showed a significant decrease in HA titer upon BPL treatment, independent of pH changes.
- Excessive M1 protein modification and loss of virion diameter were observed in H3N2 viruses treated with 0.1% BPL.
- A 0.02% BPL concentration successfully inactivated H3N2 virus without significant HA loss or structural damage.
- The 0.02% BPL was fully hydrolyzed within 7 hours at 37°C.
Conclusions:
- The sensitivity of H3N2 virus to BPL is linked to virion disruption and M1 protein modification.
- A low concentration (0.02%) of BPL provides effective inactivation of H3N2 virus while preserving HA titer and virion integrity.
- Mild BPL treatment followed by incubation offers a viable method for influenza virus inactivation in vaccine development.
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