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.

Wild-type control virus MHV-A59 and Brts31 were propagated from recombinant vaccinia virus-based infectious clones as described previously (14) . MHV-Brts105 was isolated following selection with 150 g/ml of 5-fluorouracil at 33°C for 16 h using the procedure described for Brts31 (14) . MHV-Brts105, Albts16, Wüts18, and Albts22 mutants were plaque purified twice on 17Cl-1 cells and completely sequenced to confirm the presence of the appropriate ts mutation and the absence of other changes. 17Cl-1, DBT, and L929 cells were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 5% tryptose phosphate broth (TPB; Sigma), 10% heat-inactivated fetal bovine serum (FBS; Biosera), antibiotics, and nonessential amino acids (NEAA; Invitrogen). Primary mouse embryo fibroblasts (Lonza), adipose-derived mesenchymal stem cells (Cyagen), and bone marrowderived mesenchymal stem cells (obtained from Darwin Prockop at the Health Science Center of Texas A&M University, Bryan, TX) were cultured in DMEM supplemented with 10% FBS, NEAA, and antibiotics. Virus titers were determined by plaque assay on 17Cl-1 cells.