Single‐cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses

Abstract

Microbes drive the biogeochemical cycles that fuel planet Earth, and their viruses (phages) alter microbial population structure, genome repertoire, and metabolic capacity. However, our ability to understand and quantify phage–host interactions is technique-limited. Here, we introduce phageFISH – a markedly improved geneFISH protocol that increases gene detection efficiency from 40% to > 92% and is optimized for detection and visualization of intra- and extracellular phage DNA. The application of phageFISH to characterize infection dynamics in a marine podovirus–gammaproteobacterial host model system corroborated classical metrics (qPCR, plaque assay, FVIC, DAPI) and outperformed most of them to reveal new biology. PhageFISH detected both replicating and encapsidated (intracellular and extracellular) phage DNA, while simultaneously identifying and quantifying host cells during all stages of infection. Additionally, phageFISH allowed per-cell relative measurements of phage DNA, enabling single-cell documentation of infection status (e.g. early vs late stage infections). Further, it discriminated between two waves of infection, which no other measurement could due to population-averaged signals. Together, these findings richly characterize the infection dynamics of a novel model phage–host system, and debut phageFISH as a much-needed tool for studying phage–host interactions in the laboratory, with great promise for environmental surveys and lineage-specific population ecology of free phages.

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

“…Phage bursts may occur in specific skin community structures. Future studies utilizing recently developed techniques, such as microfluidic digital PCR (Tadmor et al, 2011), phageFISH (Allers et al, 2013) and viral tagging , will provide further insight on the diversity and dynamics of the phage populations in the human microbiome and the preypredator relationship between the bacterial hosts and phages.…”

Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses

“…These include culture-based approaches (for example, targeted isolations with existing and new marine bacterial, archaeal and eukaryotic cultures), as well as new methods that either require only the host to be in culture (for example, viral tagging; Deng et al, 2012) or are completely cultivation-independent (for example, physical fractionation of viral assemblages; Bergeron et al, 2007;Steward and Rappé, 2007;Brum and Steward, 2011;Brum et al, 2013). The abundance and distribution of genetically characterized, non-tailed viruses could also be explored using phageFISH (Allers et al, 2013). Also, viruses with particular nucleic acid types can be examined by enriching for ssDNA (Kim and Bae, 2011) or specifically targeting dsDNA, ssDNA and RNA pools (Andrews-Pfannkoch et al, 2010).…”

Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans

“…Conclusively, our study provides an example of the complementary nature of culture-dependent and culture-independent approaches, and HMO-2011 will be an indispensable resource and a valuable model system for marine virus research. To understand more comprehensively the abundance, distribution, and ecological roles of this phage type in the ocean, further studies may focus on comparative analyses with more viromes from various ocean regions, functional identification of novel genes in HMO-2011, and elucidation of infection network and host-phage dynamics using techniques such as viral tagging and phageFISH (62,63).…”