Nanostructural characterization of Sf9 cells during virus‐like particles generation

Lee, Gi-Ja; Lee, Su‐Hwa; Lee, Young Ju; Quan, Fu‐Shi

doi:10.1002/sca.21322

Cited by 3 publications

(6 citation statements)

References 23 publications

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“…Spodoptera frugiperda (Sf9) cells were cultured in a serum-free SF900II medium (Invitrogen, Carlsbad, California, USA) at 27°C at a speed of 130–135 rpm. 18 Polyclonal antibodies against influenza virus were obtained by infecting mice with the influenza virus. Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (IgG) (Southern Biotech, Birmingham, AL, USA) and diaminobenzidine (DAB) (Invitrogen) were prepared for the immunostaining process.…”

Section: Methodsmentioning

confidence: 99%

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

et al. 2021

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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.

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Section: Methodsmentioning

confidence: 99%

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

et al. 2021

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“…Из указанной вируссодержащей жидкости готовили серию десятикратных разведений на питательной среде Игла МЕМ с 10% содержанием сыворотки ние значений шероховатости связано с выходом вируса из клетки и разрушением мембраны. В аналогичных исследованиях, посвященных изучению взаимодействия вирусов с клетками методами АСМ, также зарегистрированы различные изменения в морфологии мембраны [8][9][10][11][12], в том числе факт увеличения ее шероховатости [10,11]. При этом М. Moloney и соавт.…”

Section: материал и методыunclassified

“…В исследованиях G. Lee и соавт. [10] также продемонстрировано увеличение шероховатости клеточной мембраны при культивировании бакуловируса на клетках Sf9, при этом авторы предложили использовать изменения значений шероховатости мембраны клетки для оценки выхода образующихся вирусных частиц.…”

Section: материал и методыunclassified

“…Более полную характеристику взаимодействия вируса с клеткой позволяют получить методы атомно-силовой микроскопии (АСМ), которые в последние годы находят все большее применение для изучения поверхностной структуры микрообъектов: бактерий [5][6][7], вирусов и эукариотических клеток [8][9][10][11][12]. Эта технология дает возможность изучить процессы, происходящие на поверхности микрообъектов (экспрессию биомолекул, адгезивные свойства, упругость и шероховатость) при различных условиях, а также оценить и описать изменение морфофункциональных характеристик микрообъекта в целом в зависимости от влияния различных условий (изменение pH, температуры, присутствие антибиотиков и т.…”

Section: Introductionunclassified

“…[5,7]. Методы АСМ широко используются для изучения процессов взаимодействия вирусов как с эукариотическими, так и с прокариотическими клетками [6,[8][9][10][11][12].…”

Section: Introductionunclassified

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A Study of the Ultrastructure of the Surface of the Transplantable Line Vero Cells Infected With the Rabies Virus (Rabv, Lissavirus, Rhabdoviridae)

Генералов¹,

Erokhin²,

Красовская³

et al. 2017

Problems of Virology

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Characteristics of the effect of attenuated rabies virus strain «Moscow 3253» on morphological parameters of transplantable line Vero cells were studied by atomic force microscopy (AFM). Methods based on phase contrast microscopy and immunofluorescence were used to confirm the specificity of interaction and to identify the infectious activity of the rabies virus. Images of intact Vero cells and Vero cells infected with rabies virus were obtained at different periods of cultivation. The character of changes in the cell dimensions (length, width, height) and the cell membrane roughness depending on the rabies virus cultivation time was determined. During the observation period both increases and decreases in the size of the cells were recorded. The size of the infected cells exceeded that of the intact. An increase in the membrane roughness in cells exposed to rabies occurred during the entire period of observation, since the first hours of the interaction of the virus with the cell, while the intact Vero cells exhibited only minor changes in the membrane surface roughness, which were not dependent on the age of the culture. The dependence of the increase in the cell membrane roughness on the infecting dose of the rabies virus was determined. The obtained results open up the prospect of developing a methodological approach to the quantitative in vitro evaluation of the rabies virus using AFM. Changes in the cell membrane roughness appear to be the most indicative parameter for such evaluation.

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Determining the Specific Activity of Anti-Rabies Sera and Immunoglobulin Using Atomic Force Microscopy of Cell Cultures

Генералов¹,

Erokhin²,

Kuznetsov³

et al. 2021

AJMB

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Background: Mouse neutralization test is widely used to determine the level of anti-rabies antibodies, but it is labor-intensive and time consuming. Alternative methods for determining the neutralizing activity of anti-rabies sera and immunoglobulin in cell cultures are also known. Methods such as FAVN and RFFIT involve the use of fluorescent diagnostics. Determination of Cytopathic Effect (CPE) is often complicated due to features of rabies virus replication in cells. Atomic Force Microscopy (AFM) is able to detect the interaction of the virus with the cell at an early stage. Therefore, in this study, a method has been developed for determining the specific activity of anti-rabies sera and immunoglobulin using AFM of cell cultures. Methods: The method is based on the preliminary interaction of rabies virus with samples of rabies sera or immunoglobulin drug, adding the specified reaction mixture to cell culture (Vero or BHK-21), and then measuring the surface roughness of the cells using AFM. AFM was carried out in the intermittent contact mode by the mismatch method in the semi-contact mode. The results were compared with the values obtained in the mouse neutralization test. The consistency of the results obtained by both methods was evaluated by Bland-Altman method. Results: The increment in the surface roughness of the cells is a consequence of the damaging effect of the virus, which is weakened as a result of its neutralization by rabies antibodies. A dilution allowing 50% suppression of the increase in the surface roughness of cells was selected as the titer of rabies sera or immunoglobulin. In this case, the recommended range for determining the antibody titer is from 1:100 to 1:3000. Conclusion: For the first time, a new methodological approach in virology and pharmaceutical research is presented in this study. The use of the proposed methodological technique will reduce the time from 21 to 2 days to obtain results in comparison with the mouse neutralization test; also, fewer laboratory animals are required in this approach which is in agreement with 3 R Principle.

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Nanostructural characterization of Sf9 cells during virus‐like particles generation

Cited by 3 publications

References 23 publications

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

A Study of the Ultrastructure of the Surface of the Transplantable Line Vero Cells Infected With the Rabies Virus (Rabv, Lissavirus, Rhabdoviridae)

Determining the Specific Activity of Anti-Rabies Sera and Immunoglobulin Using Atomic Force Microscopy of Cell Cultures

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