Viral intra-host evolutionary dynamics revealed via serial passage of Japanese encephalitis virus in vitro

Abstract

Analyses of viral inter- and intra-host mutations could better guide the prevention and control of infectious diseases. For a long time, studies on viral evolution have focused on viral inter-host variations. Next-generation sequencing has accelerated the investigations of viral intra-host diversity. However, the theoretical basis and dynamic characteristics of viral intra-host mutations remain unknown. Here, using serial passages of the SA14-14-2 vaccine strain of Japanese encephalitis virus (JEV) as the in vitro model, the distribution characteristics of 1,788 detected intra-host single-nucleotide variations (iSNVs) and their mutated frequencies from 477 deep-sequenced samples were analyzed. Our results revealed that in adaptive (baby hamster kidney (BHK)) cells, JEV is under a nearly neutral selection pressure, and both non-synonymous and synonymous mutations represent an S-shaped growth trend over time. A higher positive selection pressure was observed in the nonadaptive (C6/36) cells, and logarithmic growth in non-synonymous iSNVs and linear growth in synonymous iSNVs were observed over time. Moreover, the mutation rates of the NS4B protein and the untranslated region (UTR) of the JEV are significantly different between BHK and C6/36 cells, suggesting that viral selection pressure is regulated by different cellular environments. In addition, no significant difference was detected in the distribution of mutated frequencies of iSNVs between BHK and C6/36 cells.

Keywords: evolutionary dynamics; intra-host single nucleotide variation (iSNV); viral intra-host evolution; viral quasispecies.

Figure 1.

The passage and sequencing strategy of JEV in BHK (A) and C6/36 (B) cells.

Figure 2.

Statistical information of iSNVs in indicated cells. (A–D) The distribution of normalized iSNVs (iSNV/kb, A), genomic coverage (B), genomic mean sequencing depth (C), and cycle threshold (Ct) values of quantitative real-time PCR (D) in BHK (left panel) and C6/36 cells (right panel) in indicated generations by smoothing regression (dashed line) with 95 per cent confidence interval (shaded area). (E) Distribution of normalized iSNVs in thirty transmission chains of BHK cells (left panel) and twenty transmission chains of C6/36 cells (right panel) by smoothing regression (dashed line) with 95 per cent confidence interval (shaded area). Statistical significance was assessed using all pairwise Kruskal–Wallis one-way ANOVA test and P-value < 0.01 is considered statistically significant.

Figure 3.

Distribution of iSNVs in BHK cells in indicated generations. (A) Distribution of the total iSNVs by smoothing regression (dashed line) with 95% confidence interval (shaded area). (B) The occurrence and development of total non-synonymous (N) and synonymous (S) iSNVs. The results are presented as the mean ± 95% confidence interval labelled by the error bars. The dashed lines represent the mean in indicated groups. (C) Distribution of total iSNVs (upper panel) and sum of normalized iSNVs (lower panel) of CDS and UTR. (D) Distribution of iSNVs at codon positions (left panel) and non-synonymous (N)/synonymous (S) iSNVs (right panel).

Figure 4.

The distribution of MuAFs of iSNVs in BHK cells. (A) The distribution of MuAFs of iSNVs along JEV genome in indicated generations. The figures of X-axis indicate the starting position of each ORF in the genome (N, non-synonymous; S, synonymous; and NC, non-coding). (B) The density distribution of the MuAFs of NC, N, and S iSNVs in indicated generations.

Figure 5.

The distribution of iSNVs and their MuAFs in C6/36 cells. (A–D) The d of iSNVs in C6/36 cells in indicated generations and detailed description information is the same as in BHK cells in Fig. 3. (E and F) The distribution of MuAFs of iSNVs in C6/36 cells and detailed description information is the same as in BHK cells in Fig. 4.

Figure 6.

The distribution of total iSNVs (upper panel) and sum of normalized iSNVs (lower panel) in each ORF of JEV in BHK (A) and C6/36 cells (B) in indicated generations. The observed normalized iSNVs for each ORF were compared with that of CDS using Mann–Whitney U test (MW) and Kolmogorov–Smirnov test (KS) with P < 0.01. Statistical significance within ORFs was assessed using all pairwise Kruskal–Wallis one-way ANOVA test with P < 0.01. The values marked without the same superscript differ significantly.