Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells

Frey, Kenneth G.; Redden, Cassie L.; Bishop-Lilly, Kimberly A.; Johnson, Reed; Hensley, Lisa E.; Raviprakash, Kanakatte; Luke, Thomas; Kochel, Tad; Mokashi, Vishwesh P.; Defang, Gabriel N.

In total, five samples were sequenced: passages 2, 6, 7, and 8 through CCL81 cells and passage 2 through MRC5 cells. All samples shared two single-nucleotide variants (SNVs) compared to the reference strain Jordan-N3/2012. Further analysis of putative SNVs indicated that all samples share an SNV at reference position 24045. This T¡C transversion falls in the S gene and results in a nonsynonymous mutation, I¡T. This variant is present in 55.8% of the reads in the MRC5 passage and 73.4% of the reads in the CCL81 passage. The proportion of reads with this SNV increased in the Vero-passaged samples, from 34.55% in passage 6 to 93.89% in passage 8, likely representing a cell culture adaptation. This residue is not predicted to reside in the binding domain of the S protein (8) . Although the reference genome contains an ambiguous nucleotide (W) at position 11262, all samples show a distinct preference for T (resulting in a leucine versus a histidine residue). It is uncertain as to whether this variation is due to a sequencing error in the reference genome or if population-level differences were lost during cell passage. Although this SNV is present in the coding sequence (CDS) of the ORF1ab gene, the functional consequences are unclear. The residue corresponding to this codon lies between the endopeptidase and the replicase domains and may not be present in mature virions. The apparent stability of this virus in vitro may facilitate the development of countermeasures by reducing the potential for rapid evolution and the resulting changes in immunodominant epitopes.