Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single-stranded DNA phage

Advances in metagenomics have revealed the ubiquity of single-stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here, we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At >= 95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 x 10(5) viruses ml(-1). These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans.

Automated summary

Advances in metagenomics have revealed the prevalence of single-stranded DNA phages of the Gokushovirinae subfamily in oceans, but little was known about their abundance and ecological roles. This study used the polony method to quantify these phages in the Gulf of Aqaba, Red Sea, and found that they were most abundant in the upper 40 meters of the water column. The study also suggested that different gokushoviruses infect bacterial hosts that are distributed differently in the water column.