Optimized Plaque Assay for Detecting Chronically Infecting Viruses of Haloarchaea

Abstract

Archaeal viruses predominantly exhibit a chronic lifestyle, where viral particles are released without causing host cell death. Therefore, conventional plaque assays, which are well-suited for identifying lytic viruses, usually fail to detect chronically infecting viruses due to their non-lytic nature. To address this limitation, we developed an optimized plaque assay protocol for detecting chronically infecting viruses of haloarchaea, particularly species within the Haloferax genus. By enhancing viral diffusibility and infectivity through adjustments in agar concentration and lowering incubation temperature, this modified protocol improved plaque formation, enabling the detection of viruses that cause mild growth delays. We demonstrate successful plaque formation for two chronically infecting viruses, Haloferax volcanii pleomorphic virus 1 (HFPV-1) and lemon-shaped virus 48N (LSV-48N), on representative Haloferax strains and highlight the potential for isolating novel viruses from environmental samples. This improved plaque assay is an effective method for the detection and quantification of chronically infecting archaeal viruses, providing a new tool for discovering new viral families in extreme environments.