Changes in ORF4 of HCoV-229E under different culture conditions

Figures

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  Fig. 1.. Alignment of ORF4 sequences in clinical specimens. The ORF4 sequence in clinical specimens was determined using Sanger sequencing and aligned with Clustal W. For comparison, the ORF4a of the laboratory strain (VR-740) and the ORF4 of the clinical strain (229E/Haiti-1/2016) were also aligned. The GenBank accession numbers for each sequence are as follows: VR-740 (OK625404), 229E/Haiti-1/2016 (MF542265), SMC-H/1121/04 (AB691764), SMC-H/1948/04 (AB691766) and SMC-H/826/04 (AB691765).

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  Fig. 2.. Analysis of ORF4 sequences in cell-adapted HCoV-229E. The ORF4 gene of HCoV-229E isolates passed in LLC-MK2 or HEF cells followed by HeLa cells was analysed using NGS. The sequencing reads were mapped to the ORF4 gene of 229E/Haiti-1/2016 (MF542265), which was used as the reference. (a–c) Mutation sites in SMC-H/1121/04, SMC-H/1948/04 and SMC-H/826/04 were visualized using Integrative Genomics Viewer, respectively. A mixture of red and blue indicates a mix of C and T bases. Black indicates a base deletion. (d) The ORF4 sequences of cell-adapted HCoV-229E strains were aligned using Clustal W. For comparison, the ORF4a of the laboratory strain VR-740 and the ORF4 of the clinical strain 229E/Haiti-1/2016 were also included in the alignment.

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  Fig. 3.. Effect of ALI culture on ORF4 gene mutations in HCoV-229E. SMC-H/1121/04 and SMC-H/1948/04, previously passaged in LLC-MK2 and HeLa cells, were used to infect HBTECs cultured under ALI conditions. The ORF4 genes of viruses collected at each time point were analysed using NGS. (a, b) Proportions of two-base deletions and nonsense mutations in SMC-H/1121/04. (c) Proportion of two-base deletions in SMC-H/1948/04. White bars indicate mutation-free reads, black bars indicate reads with two-base deletions and grey bars indicate reads with nonsense mutations.