Comparison of genome replication fidelity between SARS-CoV-2 and influenza A virus in cell culture

Calu-3 cells are susceptible to both SARS-CoV-2 and influenza A virus infection

First, we examined the growth kinetics of SARS-CoV-2 and IAV. Calu-3 cells, an adenocarcinoma cell line derived from human lung epithelial cells and susceptible to both viruses^6,7,8,9, were used for this analysis, as the tested viruses are RNA viruses that cause acute respiratory diseases. Calu-3 cells were inoculated with IAV or SARS-CoV-2 at a multiplicity of infection (MOI) of 1. The titres of the progeny viruses were measured using a plaque assay. Both viruses showed similar growth kinetics and reached a plateau at 24–36 h post infection (hpi) (Fig. 1). The virus production yields of IAV at 24 hpi and SARS-CoV-2 at 36 hpi were 1.1 × 10⁶ and 6.5 × 10⁷ pfu/mL, respectively.

Similar growth kinetics between IAV and SARS-CoV-2 in Calu-3 cells. Calu-3 cells were infected with IAV or SARS-CoV-2 at a multiplicity of infection (MOI) of 1. Culture supernatants were collected at the indicated times, and virus titres were measured by plaque assay. The bar plot represents the mean ± SD. hpi hours post infection.

Mutation rate of SARS-CoV-2 in vitro is much lower than that of IAV

To calculate the mutation rates in the genome replication of IAV and SARS-CoV-2, each virus was serially passaged every 48 h in Calu-3 cells. After 15 serial passages, culture supernatants were collected and clarified by centrifugation. Viral RNA was extracted from the supernatant after centrifugation and used for genetic analysis. For each virus, three independent lines of passages (P15-A, B, and C) were used.

The HA [1769 nucleotides (nt)] and NA (1451 nt) genes for IAV and S gene (3838 nt) for SARS-CoV-2, which encode viral surface glycoproteins, were selected for gene sequence determination. Based on the RNA samples, after 15 passages of SARS-CoV-2 (SP15-A, -B and -C) and IAV (IP15-A, -B and -C), the HA and NA genes for IAV and S genes for SARS-CoV-2 were amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and then cloned into the plasmids. The nucleotide sequences of 20 clones were determined for each RNA sample. The positions of the mutations introduced until passage 15 and the number of clones with the mutation are shown in Figs. 2, 3, and 4.

Mutations detected in IAV HA gene after 15 serial passages in Calu-3 cells. The graph shows the position and frequency of mutations in IAV HA gene. The vertical axis shows the number of clones in which mutations were observed among the 20 clones, and the horizontal axis indicates the position at which the mutation occurred. HA gene structure and protein domains are shown below the graph. Colours indicate the type of mutations: blue, synonymous mutations; red, non-synonymous mutations, black: deletions. TM transmembrane, CT cytoplasmic tail region.

Mutations detected in IAV NA gene after 15 serial passages in Calu-3 cells. The graph shows the position and frequency of mutations in NA gene. The vertical axis shows the number of clones in which mutations were observed among the 20 clones, and the horizontal axis indicates the position at which the mutation occurred. NA gene structure and protein domains are shown below the graph. Colours indicate the type of mutations: blue, synonymous mutations; red, non-synonymous mutations, black: deletions. TM transmembrane, CT cytoplasmic tail region.

Mutations detected in SARS-CoV-2 S gene after 15 serial passages in Calu-3 cells. The graph shows the position and frequency of mutations in SARS-CoV-2 S gene. The graph shows the position and frequency of mutations in S gene. The vertical axis shows the number of clones in which mutations were observed among the 20 clones, and the horizontal axis indicates the position at which the mutation occurred. S gene structure and protein domains are shown below the graph. Colours indicate the type of mutations: blue, synonymous mutations; red, non-synonymous mutations, black: deletions. SP signal peptide, NTD N-terminal domain, RBD receptor-binding domain, RBM receptor-binding motif, SD1/2 subdomain 1 and 2, FP fusion peptide, HR1 heptad repeat regions 1.

Regarding IAV, mutations were observed at 24 sites in HA and 29 sites in NA in the three passage lines after 15 passages (IP15-A, -B, and -C). The mutations were highly frequent between 650 and 730 nt in HA (Fig. 2). The mutation at 1482 bp was also dominant in IP15-A and B. In contrast, there were no common mutations among the three passage lines in the coding region of NA (Fig. 3). The only mutation site found in all three passage lines was the C to G mutation at nucleotide position 1430 within the untranslated region. The number of mutation sites in the SARS-CoV-2 S gene was 13, which was much lower than that in the IAV HA and NA genes (Fig. 4). No common mutations were observed among the three passage lines of SARS-CoV-2.

The 20 clones of IAV passage lines (IP15-A, -B, and -C) contained 76, 50, and 41 mutations in HA, and 31, 25, and 38 mutations in NA, respectively, whereas those of SARS-CoV-2 (SP15-A, -B, and -C) showed 3, 6, and 4 mutations in the S gene, respectively. The mutation rates were calculated based on this information and the data are summarized in Table 1. The average of mutation rates (mutations per nucleotide) of three passage lines after 15 passages were 1.35 × 10^–3 (± 4.07 × 10^–4) substitutions/site for IAV and 5.65 × 10^–5 (± 1.63 × 10^–5) for SARS-CoV-2. The average mutation rates per passage were 9.01 × 10^–5 (± 2.71 × 10^–5) substitutions/site/passage for IAV and 3.76 × 10^–6 (± 1.09 × 10^–6) for SARS-CoV-2, indicating that the mutation rate of SARS-CoV-2 was 23.9-fold lower than that of IAV (Fig. 5).

Number of mutations including deletions and insertions per 10⁵ nucleotides/passage. The average number of mutations after 15 serial passages of IAV and SARS-CoV-2 are shown. The vertical axis represents the number of mutations per 10⁵ nucleotides per passage. IAV nucleotides include HA and NA gene sequences, and SARS-CoV-2 refers to S gene sequences. The bar plot represents the mean ± SD. ****p < 0.0001 (Mann–Whitney-U test).

Frequencies of synonymous and non-synonymous mutations differ between IAV and SARS-CoV-2

Next, we calculated the frequencies (mutation rates) of synonymous and non-synonymous mutations in the coding regions and insertions/deletions (indels) in each viral gene. The results are presented in Table 2. The average frequencies of non-synonymous and synonymous mutations were 1.21 × 10^–3 (± 7.78 × 10^–5) and 4.02 × 10^–4 (± 5.97 × 10^–4) substitutions/site for IAV HA, 5.32 × 10^–4 (± 1.63 × 10^–4) and 5.08 × 10^–4 (± 1.08 × 10^–4) for IAV NA, and 1.31 × 10^–5 (± 0.00) and 1.31 × 10^–5 (± 1.31 × 10^–5) for SARS-CoV-2 S, respectively. The ratios of non-synonymous to synonymous mutations (dN/dS) in HA, NA, and S genes were 3.0, 1.0, and 1.0, respectively, suggesting a strong positive selection for HA.

In IAV HA, non-synonymous mutations were concentrated in the receptor-binding domain (RBD) (Fig. 2). The non-synonymous mutations A672G (amino acid position: T214A) and A709G/A709C (amino acid position: K226R/K226T) were observed in many clones, suggesting that these amino acid substitutions are involved in the acquisition of growth advantage in Calu-3 cells. In the SARS-CoV-2 S gene, synonymous and non-synonymous mutations were scattered in the gene (Fig. 4).

Mutations are mostly transitions in the SARS-CoV-2 S gene

We further analysed the mutations introduced into the HA, NA, and S genes for transition and transversion mutations. The total numbers of transitions and transversions were 133 and 121 in the IAV HA and NA genes and eight and one in the SARS-CoV-2 S gene, respectively (Fig. 6). Thus, the frequencies of transition and transversion were similar in IAV, whereas most mutations in SARS-CoV-2 were transitions.

Nucleotide substitution in IAV and SARS-CoV-2 after 15 serial passages. Nucleotide substitutions were counted in IAV and SARS-CoV-2 after 15 serial passages. IP15 and SP15 indicate 15 serial passage lines of IAV and SARS-CoV-2, respectively. Colours indicate the type of substitutions: blue, transition; white, transversion.

Indels frequencies are similar between IAV and SARS-CoV-2

The frequencies of indels in IAV HA and NA genes were 2.94 × 10^–5 (± 2.94 × 10^–5) and 7.09 × 10^–5 (± 9.38 × 10^–5) substitutions/site, respectively, and the average frequency for both genes was 4.82 × 10^–5 (± 4.25 × 10^–5) substitutions/site (Table 2). In contrast, that of the SARS-CoV-2 S gene was 1.75 × 10^–5 (± 1.52 × 10^–5) substitutions/site. There was no significant difference in the frequency of indels between IAV and SARS-CoV-2.