Experimental Evolution of HCoV-OC43 and MHV in cell culture at high and low MOI

In this study, we conducted experimental evolution experiments involving two viruses, HCoV-OC43 (ATCC VR1558) and MHV (ATCC VR766), to explore their evolutionary dynamics. These experiments were carried out in cell culture and with varying conditions of Multiplicity of Infection (MOI). We maintained three replicates for each experimental condition.Cell Culture Conditions:The maintenance medium for cells infected with HCoV-OC43 was DMEM supplemented with 0.22 percent (w/v) sodium bicarbonate (Sigma Aldrich), sodium pyruvate (Sigma Aldrich), 2 percent FBS, 1 x penicillin-streptomycin (Gibco-Invitrogen), and 1 x amphotericin B (Gibco-Invitrogen).CCL-9.1 cells infected with MHV were maintained at 37C in DMEM media supplemented with 0.22 percent (w/v) sodium bicarbonate (Sigma Aldrich), sodium pyruvate (Sigma Aldrich), 10 percent horse serum, 1x penicillin-streptomycin, 1 x amphotericin B, and non-essential amino acids.Virus Inoculum Preparation:To initiate the evolution experiments, cells were grown to 70 - 80 % confluency.After approximately 20 - 22 hours, these cell layers were infected with the respective virus at a Multiplicity of Infection (MOI) of 0.01. The incubation conditions for HCoV-OC43 were 4 days at 33 C, while MHV was grown for 20 hours at 37 C. Subsequently, supernatants from the infected cells were collected to serve as the infectious stock for the experiments. Passaging of Viruses: For successive passages, all cell types were cultured in 6-well plates until reaching 70 - 80 percent confluency. These cells were then infected with 250 microL of the undiluted viral inoculum obtained from the previous passage. The MOI was not held constant throughout all passages. At high MOI, passages were carried out without dilution to maximize the MOI, while for low MOI samples, the inocula were diluted to ensure that each cell was infected with no more than one infectious particle.Sequencing:We performed sequencing on four equidistant time series of the evolved viruses under both high and low MOI conditions (if RNA was available, considering the very low titer). Additionally, ancestral virus samples (the same sample for HCoV-OC43 in BHK-21 and HCT-8) were also sequenced. Libraries for high-throughput genome sequencing (HTS) were prepared by Novogene (UK) Company Limited (www.novogene.com). Each library was constructed using a minimum of 200 ng of total RNA, or a higher quantity if available. The total RNA samples were depleted of ribosomal RNAs (rRNA) from both eukaryotes and prokaryotes. The remaining RNAs were fragmented into 250-300 bp fragments and reverse-transcribed into double-stranded cDNAs. This was followed by end repair, A tailing, and adapter ligation. After fragment size selection and PCR amplification, the metatranscriptome libraries were rigorously quality-checked, and sequencing was executed on the Illumina platform by Novogene Company Limited (UK). Please note that the sequencing was not strand-specific.