Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

• Type of Material: Article
• Authors: Swarnendu Basak, Kyung Hee University; Hae-Ji Kang, Kyung Hee University; Ki-Back Chu, Kyung Hee University; Judy Oh, Emory University; Fu-Shi Quan, Kyung Hee University
• Language: English
• Date: 2021-01
• Publisher: SAGE
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2021
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): https://doi.org/10.1177%2F00368504211004261
• Title of Journal or Parent Work: Science Progress
• Volume: 104
• Issue: 1
• Start Page: 00368504211004261
• Grant/Funding Information: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the Ministry of Health & Welfare, Republic of Korea (HV20C0085, HV20C0142), and the National Research Foundation of Korea (NRF) (2018R1A6A1A03025124).
• Keywords: immunostaining; hemagglutinin; influenza virus; plaque assay; Baculovirus
• Research Categories: Biology, Virology; Health Sciences, Immunology

Abstract

Recombinant baculoviruses (rBVs) have been extensively used to generate virus-like particles, and baculoviruses expressing antigenic proteins have become efficient tools for inducing protective immunity. However, current methods for generating baculoviruses are costly and inefficient. Thus, the development of a simple, rapid, and accurate method of baculovirus titration is critically important. We established a method of plaque assay using an immunostaining method by which plaques can be easily visualized in Sf9 cells under a light microscope. Sf9 cells were infected with recombinant baculoviruses expressing influenza hemagglutinin surface proteins from H1N1 (A/California/04/09) or rH5N1 (A/Vietnam/1203/04). The infected cells were incubated with anti-HA antibody and the plaques were visualized using the chromogen 3′3-diaminobenzidine (DAB). Plaques were observed from days 1 to 6 post-infection, and differences in Sf9 cell seeding densities resulted in variations in the final plaque quantification. Sf9 cells seeded at a concentration of 5.5 × 104 cells/well or 7.5 × 104 cells/well showed the higher plaque titers at days 3, 4, and 5 post-infection than those found at days 1, 2, and 6 post-infection. With 5.5 × 104 cells/well or 7.5 × 104 cells/well of cell concentrations, recombinant baculovirus for rBV-HA (H1N1) showed 6 × 107 pfu/ml of titer and rBVs for rBV-HA (rH5N1) showed 5.4 × 107 pfu/ml of titer. Three days of baculovirus incubation with a certain concentration of Sf9 cells seeded are required for a rapid, simple, and accurate plaque assay, which could significantly contribute to all baculovirus-related studies.