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Quantification of diverse virus populations in the environment using the polony method


Viruses are globally abundant and extremely diverse in their genetic make-up and in the hosts they infect. Although they influence the abundance, diversity and evolution of their hosts, current methods are inadequate for gaining a quantitative understanding of their impact on these processes. Here we report the adaptation of the solid-phase single-molecule PCR polony method for the quantification of taxonomically relevant groups of diverse viruses. Using T7-like cyanophages as our model, we found the polony method to be far superior to regular quantitative PCR methods and droplet digital PCR when degenerate primers were used to encompass the group’s diversity. This method revealed that T7-like cyanophages were highly abundant in the Red Sea in spring 2013, reaching 770,000 phages ml−1, and displaying a similar depth distribution pattern to cyanobacteria. Furthermore, the abundances of two major clades within the T7-like cyanophages differed dramatically throughout the water column: clade B phages that carry the psbA photosynthesis gene and infect either Synechococcus or Prochlorococcus were at least 20-fold more abundant than clade A phages that lack psbA and infect Synechococcus hosts. Such measurements are of paramount importance for understanding virus population dynamics and the impact of viruses on different microbial taxa and for modelling viral influence on ecosystem functioning on a global scale.

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We thank K. Zhang and G. Church for help with initial set-up of the polony method in our laboratory, B. Cunningham for advice on iron chloride flocculation, Lindell laboratory members for many helpful discussions and ideas during the development of the method, I. Izhaki, G. Yahel and M. Bocharenko for advice on statistical analyses, R. Kishony for use of laboratory facilities, and the Interuniversity Institute for Marine Sciences of Eilat and the Ruppin School of Marine Sciences for access to sampling facilities and B. Mosevitzky for sample collection in September 2012. We also thank S. Avrani, O. Beja, M. Breitbart, M. Carlson, Y. Mandel-Gutfreund, G. Sabehi, D. Schwartz, D. Shitrit and J. Weitz for comments on this or an earlier version of the manuscript. This research was funded by European Council FP6 Marie Curie Reintegration grant no. 046549, Israel Science Foundation Individual grant no. 749/11, European Research Council Consolidator Grant 646868 and the Mallat Family Fund and Cullen Fund from the Technion awarded to D.L.

Author information

N.B. set up the polony method for viruses. N.B., S.G and I.M. designed, performed and analysed the experiments for method optimization and validation as well as field analyses, and contributed to writing of the manuscript. D.L. conceived the project, participated in experimental design and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Correspondence to Debbie Lindell.

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Fig. 1: Development of the polony method for viral ecology.
Fig. 2: Comparison of the polony method with other quantitative PCR methods.
Fig. 3: Analysis of T7-like cyanophages in coastal waters from the Red Sea using the polony method.
Fig. 4: Neighbour-joining tree of the DNA polymerase gene from T7-like cyanophages and sequenced polonies.
Fig. 5: Depth profiles of T7-like cyanophages and cyanobacteria collected from the Gulf of Aqaba, Red Sea during the spring bloom on 4 April 2013.