Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants

Abstract

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named “Cedratvirus.” The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.

Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses

“…The isolation of pacmanvirus confirms that the coculture strategy on protists in association with flow cytometry (8,30) detection from culture supernatant is an extremely fast, sensitive, and simple way to detect new viruses. Considering the recent expansion of newly described giant amoeba viruses, it seems difficult to define and apply the concept of a new family for pacmanvirus.…”

Cedratvirus, a Double-Cork Structured Giant Virus, is a Distant Relative of Pithoviruses

“…Once detected, and in the case of viral mixture, the end point dilution and centrifugation methods failed to separate and purify each viral population. Therefore, we applied a new sorting technique adapted in our lab and under review to purify Cedratvirus by flow cytometry using a BD FACS JAZZ ® sorter (BD biosciences, Rungis, France) [13]. Purity control was assessed using electron microscopy.…”

Advantages and limits of metagenomic assembly and binning of a giant virus

“…Sample was then processed by flow cytometry for sorting using the BD FACSAria™ Fusion cell sorter cytometer (BD Biosciences). After determining 40 populations, sorting was performed in 96 well microplates as previously described (Khalil et al, 2016a) . Co-cultivations were then performed on the sorted samples using Acanthamoeba castellanii strain Neff and Vermamoeba vermiformis as cell hosts, with 10 microplates for each host.…”