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.

Competitive fitness assays. Continuous 17Cl-1 cells or primary MEFs (mouse embryo fibroblasts) were inoculated with a mixed virus pool containing 0.005 PFU per cell each of wild-type virus and one ts mutant. Cells were incubated at 33°C for 24 h on 17Cl-1 or 48 h on MEFs to allow the viruses to complete several rounds of replication. Relative amounts of wild-type and mutant virus were quantified either by RT-PCR following the method of Hall and Little (39) or by two replicate plaque assays, of which both were inoculated at 33°C for 1 h and then one was incubated at 33°C and the other at 40°C for 3 days. For RT-PCR quantification, released virus was concentrated by precipitation with 10% polyethylene glycol 8000 and purified by pelleting through a sucrose-TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA) cushion (25% [wt/vol] sucrose in 50 mM Tris-HCl [pH 7.4], 100 mM NaCl, and 0.1 mM EDTA). RNA was extracted from purified virus using an RNeasy minikit (Qiagen). Purified wild-type and Brts105 RNAs were mixed in molar ratios from 1:5 to 5:1 for use as standards at this stage. Virion RNA and standards were converted to cDNA by reverse transcription with random hexamer primers and amplified with primers bracketing the mutation in Brts105, forward primer 5= GCAATAGGTATGATGTGTG 3= and reverse primer 5= GCTGTGGT TGCCGTATTGTAAG 3=. Amplicons were then sequenced commercially (Source Bioscience) using the same MHV-specific primers. Results were regressed to the proportion of sequencing peak height, normalized to standards.