# Illuminating the newly produced viruses within the virosphere with bioorthogonal noncanonical amino acid tagging and single-virus genomic sequencing technologies

## TL;DR

This study introduces a new method to identify and analyze active viruses in marine environments by labeling and sequencing newly produced viral particles.

## Contribution

The paper introduces viral BONCAT-FACS, a novel method combining bioorthogonal tagging and flow cytometry to track active viral infections in natural environments.

## Key findings

- BONCAT-FACS successfully labeled and sorted diverse marine bacteria, microeukaryotic cells, and virioplankton.
- Active virocells showed a strong enrichment in viral contigs compared to inactive cells.
- The method revealed distinct turnover patterns in specific virus groups like pelagiphages and NCLDV viruses.

## Abstract

Marine viruses impact biogeochemical cycles through cell lysis, releasing organic matter and nutrients that fuel ocean productivity. Identifying and quantifying the specific viruses active in these processes remain a priority in the field. Here, we introduce a click-chemistry method to fluorescently label, sort, and sequence the genomes of newly produced viral particles (viral progeny) released from transcriptionally active host microbial cells, alongside the analysis of co-occurring inactive cells and pre-existing viruses in environmental samples. This approach, called viral bioorthogonal noncanonical amino acid tagging (BONCAT)-fluorescence-activated cell sorting (FACS), combines BONCAT with environmental sample incubation, followed by single-virus and single-cell sorting by flow cytometry (FACS). Genomic analysis of translationally active cells and new viral progeny in coastal seawater incubations confirmed BONCAT labeling and successful sorting of diverse marine bacteria, microeukaryotic cells, and virioplankton, with stark differences in the predicted turnover of specific groups of infecting viruses, including pelagiphages, methylophages, a Flavobacteriales-associated novel “Far-T4” clade, noncanonical DNA viruses of Naomiviridae using dU instead of dT, algae-infecting giant NCLDV viruses, and parasitic virophages. Sequenced BONCAT-active cells showed a strong enrichment in viral contigs relative to the inactive cell fraction, suggestive of a large proportion of translationally active virocells. This study illustrates the effectiveness of viral BONCAT-FACS for uncovering genome-resolved virus–host dynamics. By providing a direct approach for tracking active viral infections in natural environments, this method enhances our ability to investigate behavior and interactions of these nanoscale predators, expanding our understanding of their role in ecosystem dynamics.