Detecting chronically infecting viruses of haloarchaea

The study of archaeal viruses is important for understanding microbial life in extreme environments. However, this study is difficult mainly because they predominantly exhibit a chronic lifestyle, where viral particles are released without causing host cell death. Therefore, conventional plaque assays, which are well-suited for studying lytic viruses, usually fail to detect chronically infecting viruses due to their nonlytic nature. To address this limitation, we developed an optimized plaque assay protocol for detecting chronically infecting viruses of haloarchaea, focusing on species within the Haloferax genus. By enhancing viral diffusibility and infectivity through adjustments in agar concentration and incubation temperature, this modified protocol improved plaque formation, enabling the detection of viruses that cause mild growth delays. We successfully demonstrate plaque formation for two chronically infecting viruses, Haloferax volcanii pleomorphic virus 1 (HFPV-1) and lemon-shaped virus of Haloferax strain Atlit 48 N (LSV-48 N), on representative Haloferax strains. This 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. Here, we present a high-sensitivity plaque assay protocol tailored specifically to detect archaeal viruses that produce chronic productive infections, which traditional methods have failed to identify. Our findings offer a novel tool that can be adapted for studying virus-host interactions within extreme environments, potentially expanding our understanding of the ecological roles and diversity of archaeal viruses. This protocol also represents a valuable advancement for microbiologists seeking to discover new archaeal viral viruses.