Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR

Abstract

Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host’s genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.

Evolution of virus and virophage facilitates persistence in a tripartite microbial system

“…For all ddPCR assays, DNA was extracted using a commercial kit (DNeasy 96 Blood & Tissue Kit, Qiagen, Hilden, Germany) and kept at 4°C until measured or measured right after sampling. We designed and selected primer and probes for virus amplification by standard PCR procedure (51). PCR parameters correspond to 1 cycle of 95°C (10 min), 40 cycle of 94°C (30 s), 40 cycle of 58°C (1 min), 1 cycle of 98°C (10 min) and hold temperature 12°C.…”

“…To propagate and produce virus stocks of selected viruses, we added 50 μL of the medium used to recover viruses from the 0.2 μm filter to medium containing the ancestral host (strain E4-10P; 10 5 cells/mL). After observing host lysis (confirmed by sampling and enumeration by microscopy), we collected virus samples (filtration through 0.45-μm filters) and quantified virus concentrations by ddPCR (51)). To produce selected virophages, we added 50 μL of the filtrate of the 0.2 μm filter to medium containing strain host (strain E4-10P; 10 5 cells/mL) and ancestral virus.…”

“…Lateral light scatter (SSC) and SYBR-green fluorescence (FITC) measurements were performed with logarithmic amplification using FlowJo software (BDBiosciences). Virus and virophage samples were immediately collected for DNA extraction and quantified by ddPCR (see above and (51)).…”

Advances in rapid point-of-care virus testing

“…The results suggest that ddPCR can be an effective method for quantifying viruses and virion phages, and we discuss the potential application of this method in studying the ecology and evolution of viruses and virion phages. 40 Also based on the ddPCR technique, Olivero et al developed an assay for the quantification of hepatitis D virus (HDV)-RNA viremia and compared the performance associated with the real-time PCR method. ddPCR provides an absolute quantitative method to standardize HDV-RNA measurements, thereby improving the clinical and diagnostic management of delta hepatitis.…”

Updated Virophage Taxonomy and Distinction from Polinton-like Viruses

“…While these data point to additional hosts of virophages, such associations would have to be confirmed experimentally, as virophages could also be detected in single-cell genomes if they were attached to or engulfed in but not infecting the protist cell. Building on these candidate associations, further characterization of virophage-host interaction would ideally be performed using recently developed in vitro assays such as virusFISH [34] and droplet digital PCR [35].…”