An optimized method for counting viral particles using electron microscopy
Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. When studying samples containing viruses, one confronts an unavoidable question of the quantitative determination of viral particles in the sample. One of the simplest and efficient...
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| Format: | Article |
| Language: | English |
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Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders
2019-05-01
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| Series: | Вавиловский журнал генетики и селекции |
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| Online Access: | https://vavilov.elpub.ru/jour/article/view/2026 |
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| author | B. N. Zaitsev O. S. Taranov N. B. Rudometova N. S. Shcherbakova A. A. Ilyichev L. I. Karpenko |
| author_facet | B. N. Zaitsev O. S. Taranov N. B. Rudometova N. S. Shcherbakova A. A. Ilyichev L. I. Karpenko |
| author_sort | B. N. Zaitsev |
| collection | DOAJ |
| description | Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. When studying samples containing viruses, one confronts an unavoidable question of the quantitative determination of viral particles in the sample. One of the simplest and efficient approaches to quantitative determination of viral particles in preparation includes the use of electron microscopy; however, a high detection threshold is a significant limitation of this method (107 particles per ml). Usually, such sensitivity is insufficient and can result in error diagnosis. This study aims to develop a method making it possible to detect the number of viral particles more precisely and work with samples in which the concentration of particles is lower than 107/ml. The method includes a concentration of viral particles on the polyethersulfone membrane applied in centrifugal concentrators and subsequent calculation using an electron microscope. We selected env-pseudoviruses using a lentiviral system making it possible to obtain standardized samples of virus-like particles that are safer than a live virus. Suspension of viral particles (a volume of 20 ml) was placed into the centrifugal concentrator and centrifuged. After that, we took a membrane out of the centrifugal concentrator and evaluated the number of particles on the ultrathin section using an electron microscope. The number of viral particles on the whole surface of the filter (a square of 4 сm2) was 4×107 virions, the initial concentration of pseudoviruses in the sample was 2×106 per 1 ml (4×107 particles per 20 ml). As a result, the developed method enables one to evade the major disadvantage of quantitative determination of viruses using electron microscopy regarding a high detection threshold (concentration of particles 107/ml). Furthermore, the centrifugal concentrator makes it possible to sequentially drift a considerable volume of the suspension through the filter resulting in enhancement of test sensitivity. The developed approach results in increased sensitivity, accuracy, and reproducibility of quantitative analysis of various samples containing animal, plant or human viruses using electron microscopy. |
| format | Article |
| id | doaj-art-308af56d1db1455fa0263a1237ad5179 |
| institution | Kabale University |
| issn | 2500-3259 |
| language | English |
| publishDate | 2019-05-01 |
| publisher | Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders |
| record_format | Article |
| series | Вавиловский журнал генетики и селекции |
| spelling | doaj-art-308af56d1db1455fa0263a1237ad51792025-02-01T09:58:07ZengSiberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and BreedersВавиловский журнал генетики и селекции2500-32592019-05-0123333734210.18699/VJ19.498912An optimized method for counting viral particles using electron microscopyB. N. Zaitsev0O. S. Taranov1N. B. Rudometova2N. S. Shcherbakova3A. A. Ilyichev4L. I. Karpenko5State Research Center of Virology and Biotechnology “Vector”State Research Center of Virology and Biotechnology “Vector”State Research Center of Virology and Biotechnology “Vector”State Research Center of Virology and Biotechnology “Vector”State Research Center of Virology and Biotechnology “Vector”State Research Center of Virology and Biotechnology “Vector”Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. When studying samples containing viruses, one confronts an unavoidable question of the quantitative determination of viral particles in the sample. One of the simplest and efficient approaches to quantitative determination of viral particles in preparation includes the use of electron microscopy; however, a high detection threshold is a significant limitation of this method (107 particles per ml). Usually, such sensitivity is insufficient and can result in error diagnosis. This study aims to develop a method making it possible to detect the number of viral particles more precisely and work with samples in which the concentration of particles is lower than 107/ml. The method includes a concentration of viral particles on the polyethersulfone membrane applied in centrifugal concentrators and subsequent calculation using an electron microscope. We selected env-pseudoviruses using a lentiviral system making it possible to obtain standardized samples of virus-like particles that are safer than a live virus. Suspension of viral particles (a volume of 20 ml) was placed into the centrifugal concentrator and centrifuged. After that, we took a membrane out of the centrifugal concentrator and evaluated the number of particles on the ultrathin section using an electron microscope. The number of viral particles on the whole surface of the filter (a square of 4 сm2) was 4×107 virions, the initial concentration of pseudoviruses in the sample was 2×106 per 1 ml (4×107 particles per 20 ml). As a result, the developed method enables one to evade the major disadvantage of quantitative determination of viruses using electron microscopy regarding a high detection threshold (concentration of particles 107/ml). Furthermore, the centrifugal concentrator makes it possible to sequentially drift a considerable volume of the suspension through the filter resulting in enhancement of test sensitivity. The developed approach results in increased sensitivity, accuracy, and reproducibility of quantitative analysis of various samples containing animal, plant or human viruses using electron microscopy.https://vavilov.elpub.ru/jour/article/view/2026electron microscopypseudovirusesconcentratingnumber of viral particles |
| spellingShingle | B. N. Zaitsev O. S. Taranov N. B. Rudometova N. S. Shcherbakova A. A. Ilyichev L. I. Karpenko An optimized method for counting viral particles using electron microscopy Вавиловский журнал генетики и селекции electron microscopy pseudoviruses concentrating number of viral particles |
| title | An optimized method for counting viral particles using electron microscopy |
| title_full | An optimized method for counting viral particles using electron microscopy |
| title_fullStr | An optimized method for counting viral particles using electron microscopy |
| title_full_unstemmed | An optimized method for counting viral particles using electron microscopy |
| title_short | An optimized method for counting viral particles using electron microscopy |
| title_sort | optimized method for counting viral particles using electron microscopy |
| topic | electron microscopy pseudoviruses concentrating number of viral particles |
| url | https://vavilov.elpub.ru/jour/article/view/2026 |
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