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. 2019 Jun 19;2:216.
doi: 10.1038/s42003-019-0475-6. eCollection 2019.

High-throughput Isolation of Giant Viruses Using High-Content Screening

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Free PMC article

High-throughput Isolation of Giant Viruses Using High-Content Screening

Rania Francis et al. Commun Biol. .
Free PMC article

Abstract

The race to discover and isolate giant viruses began 15 years ago. Metagenomics is counterbalancing coculture, with the detection of giant virus genomes becoming faster as sequencing technologies develop. Since the discovery of giant viruses, many efforts have been made to improve methods for coculturing amebas and giant viruses, which remains the key engine of isolation of these microorganisms. However, these techniques still lack the proper tools for high-speed detection. In this paper, we present advances in the isolation of giant viruses. A new strategy was developed using a high-throughput microscope for real-time monitoring of cocultures using optimized algorithms targeting infected amebas. After validating the strategy, we adapted a new tabletop scanning electron microscope for high-speed identification of giant viruses directly from culture. The speed and isolation rate of this strategy has raised the coculture to almost the same level as sequencing techniques in terms of detection speed and sensitivity.

Keywords: Virus-host interactions; Water microbiology.

Conflict of interest statement

Competing interestsThe authors declare no competing non-financial interests but the following competing financial interests: Y.O. is an employee at Hitachi High Technologies.

Figures

Fig. 1
Fig. 1
Giant virus infectivity profiles and signatures in A. castellanii Neff analyzed by high-content screening. Targeted algorithms for image analysis were configured on the negative control A. castellanii Neff stained with SYBR Green. The mean values are represented for each parameter (n = 3 independent experiments). Error bars represent standard deviations. In addition, p-values were generated for each parameter. a Cell Shape P2A Index (p-value of 0.02). b SYBR Green Average Intensity p-value of 0.008 and c total cell count (p-value of 0.005)
Fig. 2
Fig. 2
Specific feature detection of infected A. castellanii Neff by high-content screening. All cells are stained with SYBR Green. The scale bars indicate 100 µm. a SYBR Green channel for APMV at 24 h pi. b Brightfield channel for APMV at 24 h pi. c, d higher magnification of APMV at 24 h pi (scale bar indicates 50 µm). Bright spots representing the viral factory (vf) are well differentiated from the nucleus (n) and the vacuoles (v). e, f Marseillevirus T19 at 10 h pi. g, h Pandoravirus massiliensis at 18 h pi. i, j Tupanvirus Deep Ocean at 24 h pi. k, l Pacmanvirus at 6 h pi. m, n Cedratvirus at 20 h pi. o, p Faustovirus E12 at 48 h pi. q, r Orpheovirus IHUMI - LCC2 at 48 h pi. s, t negative control A. castellanii Neff at 48 h pi at high-magnification (scale bar indicates 50 µm)
Fig. 3
Fig. 3
Correlation between fluorescence signal intensity increase and viral DNA replication in A. castellanii Neff infected with a APMV, b Marseillevirus, c Pandoravirus, d Tupanvirus, e Pacmanvirus, f Cedratvirus, g Faustovirus and h Orpheovirus. The mean values and the standard deviations (n = 3 independent experiments) are represented (p-value of 0.009)
Fig. 4
Fig. 4
SEM images of culture supernatants showing some of the isolated giant viruses. These photos are generated from our samples using the TM4000 Plus microscope. a Uninfected A. castellanii Neff (red arrow indicates nucleus). b Mimivirus particles showing a typical ~ 650 nm capsid (red arrows). c, d Pandoravirus particles with their characteristic apical aspect. e A. castellanii Neff cell with Tupanvirus particles adhered to its surface (red arrows). f High-magnification image of e showing typical Tupanvirus particles with their characteristic tails (red arrows). g, h Supernatant of an infected culture showing clusters of Marseillevirus particles with a ~250 nm capsid (red arrows indicate clustered particles). Scale bar and acquisition settings are generated automatically by the SEM on the original micrographs
Fig. 5
Fig. 5
Historic evolution of giant virus isolation strategies since their discovery. Until 2013, giant virus isolation was performed by traditional and operator-dependent techniques using optical microscopy and staining. In 2016, flow cytometry introduced the concept of automated detection and identification. Here, we developed a new isolation strategy introducing new tools allowing the live monitoring of cocultures and high-content analysis for a rapid detection and identification of giant viruses

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