An optimized method for counting viral particles using electron microscopy

Abstract

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.