Fig 1.
Experimental design of the in vitro generation of genetic variability studies for HSV-1 and HSV-2 subtypes and detected changes in plaque phenotype.
(A) Five viral clones from each original stock were five times plaque-purified in Vero cells and then deep sequenced. Two clones were re-sequenced at ultrahigh depth, whose replication, infectivity, and pathogenesis were compared to their corresponding parental stocks in cell culture and animal models of infection (S4 Fig). Those two clones were used to infect Vero and HaCaT cells at an MOI of 0.1 PFU/cell. After 48 hpi, viral progenies were harvested, referring to this infection cycle as a passage. Viral populations from each plaque-purified clone were ultra-deep sequenced after five and ten passages in each cell line. (B) Four representative pictures are showing the plaque morphology phenotype of HSV-1 clone 3 in Vero cells (48 hpi), before (passage 0, P0), and after ten passages (P10) in Vero cells. This syncytial plaque phenotype is due to the previously well-described syncytia-inducing mutation in UL27 CDS (see Sheet I in S1 Table, variant #38: R858H) [44]. Tiled images (4 x 4) were taken using a Leica DM IL LED inverted microscope equipped with a Leica DFC3000-G digital camera. Scale bars indicate 100 μm.
Fig 2.
Genetic diversity in viral populations from original stocks and five times plaque-isolated HSV-1 and HSV-2 clones.
(A) Total number of MVs observed in each original stock, at a frequency equals or above a 1% limit of detection (see Material and Methods for details). The total number of MVs (y-axis) is separated into variant type and genomic location (x-axis). Variant type distinguishes between SNPs and InDels, discriminating nonsynonymous SNPs. The genomic location of each variant is categorized as non-coding or coding region. (B) Histograms show the number of MVs in each frequency range for each original stock. The frequency of each variant was examined and grouped in shown ranges (e.g., 10% to <20% frequency, 20% to <30% frequency and so on). SNPs and InDels were combined for this analysis. (C) Total number of MVs observed in each HSV-1 (left, blue bars) and HSV-2 (right, red bars) clones after five rounds of plaque-isolation, categorized by nonconservative changes (SNPs and InDels). See Sheets C and D in S1 Table for full lists of MVs position and frequency data. (D) Total number of variants detected in each clone are grouped by subtype and graphed showing mean +/- SEM (ns = not significant p > 0.05 by two-tailed Mann–Whitney U-test).
Fig 3.
Comparative variant analysis from standard- and ultra-deep sequencing data of two plaque-isolated HSV-1 and HSV-2 clones.
(A) Histograms show the average depth of coverage per genomic position of reads alignments from standard-deep sequencing (SDS, white bars) and ultra-deep sequencing (UDS, black bars) for each viral clone. (B) Histograms bar plot total number of MVs observed after variant analysis of SDS and UDS data in each viral clone, discriminating between preexisting MVs found in the corresponding original stock (white) and de novo appearance (red). (C) The number of total, preexisting, and de novo MVs detected in each clone from SDS, and UDS data are grouped and graphed (blue shapes for HSV-1, red for HSV-2 clones), showing mean +/- SEM (ns p > 0.05, * p = 0.029 by two-tailed Mann–Whitney U-test). (D) Number of de novo MVs observed after variant analysis of SDS and UDS data in each viral clone, stacked by frequency ranges. SNPs and InDels were combined for this analysis. (E) de novo MVs with a frequency between 1% to 2% detected in each clone from SDS, and UDS data are grouped and graphed (blue shapes for HSV-1, red for HSV-2 clones), showing mean +/- SEM (* p = 0.029 by two-tailed Mann–Whitney U-test). See Sheets E and F in S1 Table for full lists of MVs position and frequency data.
Fig 4.
Comparison of de novo generation of total and nonconservative genetic diversity between HSV-1 and HSV-2 purified clones, after five (P5) and ten (P10) passages in Vero and HaCaT cells.
(A) Total number of MVs are plotted according to variant analysis data for each clone, passage, and cell line, differentiating between preexisting MVs observed in the corresponding passage zero (P0) in white, and de novo generated MVs in red. (B) Number of de novo SNPs detected in each viral population, categorized by nonsynonymous (black) and synonymous/non-coding changes (white). (C) Number of de novo InDels detected among each viral population are stacked by their location impacting coding regions (black) or non-coding regions (white). (D) De novo MVs are stacked by frequency ranges. SNPs and InDels were combined for this analysis. See Sheets G-N in S1 Table for full lists of MVs position and frequency data.
Fig 5.
Stacked histograms show the number of de novo genic MVs (x-axis) located in each HSV-1 (left) and HSV-2 (right) coding sequence (gene; y-axis), after five (P5) and ten (P10) passages in Vero and HaCaT cells. Only coding sequences registering at least one variant are included in the histogram. MVs found in both copies of each RL1, RL2, and RS1 coding sequences are listed together. SNPs and InDels were combined for this analysis. See Sheets G-N in S1 Table for full lists of MVs position and frequency data.
Fig 6.
Dynamics of nonconservative de novo variants in each HSV-1 (left) and HSV-2 purified populations (right), whose frequency increased over sequential passages in cell culture. Nonsynonymous de novo SNPs were plotted by their frequency in the sequenced viral population after five (P5) and ten (P10) passages in Vero and HaCaT cells. SNPs, their encoded proteins (bold), as well as the change that they would cause in the translated protein (italic), are listed in the legend according to their frequency at P5 and P10.