Competitive Fitness in Coronaviruses Is Not Correlated with Size or Number of Double-Membrane Vesicles under Reduced-Temperature Growth Conditions

Al-Mulla, Hawaa M. N.; Turrell, Lauren; Smith, Nicola M.; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C.; Siddell, Stuart G.; Neuman, Benjamin W.

Functional characterization of ts viruses. To better understand the functional consequences of activating the ts phenotype of Brts105, we performed a stop/resume assay, which was designed as a simple tool to compare the severity of ts phenotypes. The assay takes advantage of the eclipse phase, during which RNA replication takes place but no new virus emerges, which lasts for 6 h after inoculation (5) . The stop/resume assay works by stopping RNA synthesis 4.5 h after inoculation, during the eclipse phase. After 3 h under stopped conditions, RNA synthesis was then either resumed for 4 h, in order to allow the release of new virus with the participation of previously synthesized components, or kept under stopped conditions (Fig. 2D ). If the resumed titer was significantly higher than the stopped titer (typically about a 10-fold increase), then the ts phenotype was classified as mild, meaning that some residual function or functional components were available after the stoppage period, but if the titer did not increase significantly after resumption, then the ts phenotype was classified as severe.