Concentration of viruses and electron microscopy

Abstract

Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Viruses are often studied by electron microscopy (EM), which provides new high-resolution data on the structure of viral particles relevant to both fundamental virology and practical pharmaceutical nanobiotechnology. Electron microscopy is also applied to ecological studies to detect viruses in the environment, to analysis of technological processes in the production of vaccines and other biotechnological components, and to diagnostics. Despite the advances in more sensitive methods, electron microscopy is still in active use for diagnostics. The main advantage of EM is the lack of specificity to any group of viruses, which allows working with unknown materials. However, the main limitation of the method is the relatively high detection limit (107 particles/mL), requiring viral material to be concentrated. There is no most effective universal method to concentrate viruses. Various combinations of methods and approaches are used depending on the virus and the goal. A modern virus concentration protocol involves precipitation, centrifugation, filtration, and chromatography. Here we describe the main concentrating techniques exemplified for different viruses. Effective elution techniques are required to disrupt the bonds between filter media and viruses in order to increase recovery. The paper reviews studies on unique traps, magnetic beads, and composite polyaniline and carbon nanotubes, including those of changeable size to concentrate viral particles. It also describes centrifugal concentrators to concentrate viruses on a polyethersulfone membrane. Our review suggests that the method to concentrate viruses and other nanoparticles should be chosen with regard to objectives of the study and the equipment status of the laboratory.

Progress in etiological diagnosis of viral meningitis

“…Transmission electron microscopy (EM) is a method that determines the pathogens by examining the cellular morphology of specimens. It lacks specificity for any specific group of viruses, which broadens the clinical outcomes and delays treatment ( 26 , 27 ). The main limitation of this method is its relatively high detection limit (10 6 –10 7 particles/mL) ( 28 ), requiring virus material concentration.…”

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

“…Метод электронной микроскопии позволяет получать данные относительно размеров, формы вирусов, их расположения в клетке, а также внутриклеточных изменений, возникающих в результате инфицирования вирусом. Электронная микроскопия позволяет работать с неидентифицированным материалом, а имеющиеся комплексные подходы по очистке и концентрированию вируссодержащих суспензий [10] открывают возможность применения метода не только для фундаментальных исследований, но и для производственных задач [11]. В качестве основных факторов, ограничивающих активное применение электронной микроскопии на контрольных этапах производства АИГ, следует указать высокую стоимость оборудования и необходимость в специализированном помещении для работы с электронным микроскопом.…”

“…Данный способ позволяет оценить количество вируса в бляшкообразующих единицах (БОЕ) 10 . Указанный метод не нашел широкого практического применения в силу своих недостатков -длительности и трудоемкости, в особенности при анализе материала с высоким содержанием вируса 11 . Применение метода бляшек также ограничено способностью штамма ВБ оказывать цитопатическое действие на клеточную культуру, проявляющееся в деструктивных изменениях отдельных клеток или всего клеточного монослоя, наблюдаемых с применением световой микроскопии.…”

Concentration of SARS-CoV-2-Infected Cell Culture Supernatants for Detection of Virus-like Particles by Scanning Electron Microscopy

“…Such samples that can be difficult to examine include the supernatants of virus-infected cells [18] as sometimes only a few virus particles are released in the culture medium upon infection. Resin-embedding of the cultured infected cells and ultramicrotomy can address this issue [19], but for more rapid TEM examination, several strategies may be used to concentrate virus-infected cell supernatants [7,20]. First, the sample can be concentrated prior to being deposited on the TEM grid, by purification and/or ultracentrifugation [20].…”

“…Resin-embedding of the cultured infected cells and ultramicrotomy can address this issue [19], but for more rapid TEM examination, several strategies may be used to concentrate virus-infected cell supernatants [7,20]. First, the sample can be concentrated prior to being deposited on the TEM grid, by purification and/or ultracentrifugation [20]. Secondly, this concentration can be performed on the TEM grid membrane during the sample deposit by (i) using the glow-discharge of the TEM grid to increase electrostatic charges and thus interactions between the virus particles and the membrane [21]; (ii) performing ultracentrifugation directly on the grid membrane with a dedicated system [22]; and (iii) coating the grid membrane with molecules or antibodies to improve adhesion of the viruses to the membrane [23,24].…”