A simple and efficient method for concentration of ocean viruses by chemical flocculation - PubMed (original) (raw)

A simple and efficient method for concentration of ocean viruses by chemical flocculation

Seth G John et al. Environ Microbiol Rep. 2011 Apr.

Free PMC article

Abstract

Ocean viruses alter ecosystems through host mortality, horizontal gene transfer and by facilitating remineralization of limiting nutrients. However, the study of wild viral populations is limited by inefficient and unreliable concentration techniques. Here, we develop a new technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research.

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Figures

Fig. 1

Fig. 1

Optimization of virus concentration and redissolution from Biosphere 2 Ocean viral-fraction seawater.A. The effect of various filters on Fe-virus concentrate recovery after flocculation with 1 mg l−1 Fe: PC = 0.8 µm polycarbonate filters (Whatman Nuclepore), PES = 0.8 µm polyethersulfone (Pall Supor), MCE = 1.2 µm mixed cellulose ester (Millipore RAWP), and GF/B = 1.0 µm nominal pore size glass fibre filters (Whatman).B. The effect of Fe addition on Fe-virus concentrate recovery by filtration onto a polycarbonate membrane or settling.C. The effect of pH and resuspension buffer on the time required for dissolution of the iron hydroxide flocculate. Resuspension buffers were tested with 0.2 M EDTA in all solutions and the addition of either 0.1 M ascorbate or 0.1 M oxalate to two treatments. One millilitre of buffer was used to dissolve 1 mg of Fe.

Fig. 2

Fig. 2

Comparison of viral concentration methods showing the experimental design schematic and resulting concentration efficiency using viral-fraction (< 0.22 µm filtrate) natural seawater from Scripps Pier in San Diego, CA. Recovery is based on virus counts by epifluorescence microscopy.

Fig. 3

Fig. 3

Phylogenetic tree of previously published gene 20 (myovirus portal protein gene) sequences and gene sequences obtained by PCR amplification of gene 20 from Fe-virus concentrates collected at Scripps Pier. Gene sequences obtained in this study are designated as ‘FeCl3’.

Fig. 4

Fig. 4

Infectivity of FeCl3-flocculated viruses after variable durations of storage (24 h to 38 days) and with different resuspension buffers. Infectivity was assessed by agar overlay plaque assay of flocculated and resuspended virus. Recovery was determined for (A) myovirus resuspended in ascorbate buffer, (B) myovirus resuspended in oxalate buffer and immediately transferred to modified SM buffer for long term storage, (C) myovirus resuspended in oxalate buffer, and (D) siphovirus resuspended in oxalate buffer. Viruses in (A) and (B) were spiked into artificial seawater prior to concentration, while viruses in (C) and (D) were spiked into aged natural seawater prior to concentration.

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References

    1. Adams MH. Bacteriophages. New York, USA: Interscience Publishers; 1959.
    1. Anderson MA, Morel FMM. The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thalassiosira weisflogii. Limnol Oceanogr. 1982;27:789–813.
    1. Angly FE, Felts B, Breitbart M, Salamon P, Edwards RA, Carlson C, et al. The marine viromes of four oceanic regions. PLoS Biol. 2006;4:2121–2131. - PMC - PubMed
    1. Bergh O, Borsheim KY, Bratbak G, Heldal M. High abundance of viruses found in aquatic environments. Nature. 1989;340:467–468. - PubMed
    1. Borrego JJ, Cornax R, Preston DR, Farrah SR, McElhaney B, Bitton G. Development and application of new positively charged filters for recovery of bacteriophages from water. Appl Environ Microbiol. 1991;57:1218–1222. - PMC - PubMed

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