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.

Quantitative reverse transcriptase PCR (qRT-PCR). Duplicate flasks were prepared so that RNA extraction and DMV analysis could be performed on samples from the same experiment. Total cellular RNA was extracted from infected cells 10 h after inoculation using an RNeasy minikit (Qiagen). The RNA integrity was analyzed with the 2100 Bioanalyzer using the RNA 6000 Nano kit (Agilent). Both cDNA synthesis and PCR were performed in a single tube from total RNA using the SensiMix SYBR one-step kit (Bioline). Genomic RNA was quantified by amplifying a 124-bp region from the nsp3 gene with the forward primer 5= AATAAG CAGGAGGCAAATGTTC 3= and the reverse primer 5= CCCACAACAC AATGAAACAAATC 3=. A 110-nt amplicon spanning the leader-body junction of sgRNA7 was amplified using forward primer 5= GTACCCTC TCAACTCTAAAAC 3= and reverse primer 5= GCGGTTTACAGAGGAG CTT 3=. Amplifications were carried out in triplicate, with a 250 nM primer concentration in 25-l reaction volumes. Melt curve analysis was performed to confirm PCR product specificity. Absolute quantitation was performed by comparison to a standard curve of cloned amplicons.