Genotypic and Phenotypic Diversity of Herpes Simplex Virus 2 within the Infected Neonatal Population

Increased plaque size in culture is not determined by viral entry, DNA replication, protein expression, or infectious virus production. Viral growth characteristics were compared for representative neonatal isolates, including large-plaque formers (green) and small-plaque formers (black). (A and B) Viral entry kinetics. (A) Viral isolates were applied to Vero cell monolayers at 4°C for 1 h to allow virus binding and were then moved to 37°C to allow virus entry. Extracellular virus was inactivated by a low-pH buffer at the times indicated (orange arrowheads). Cell monolayers were washed and overlaid with methylcellulose. Plaques were scored after 100 h of incubation. PBS, phosphate-buffered saline. (B) Viral entry was quantified as the fraction of plaques formed following citrate buffer application, where 100% represents the number of plaques formed on a monolayer not treated with citrate buffer (control). These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (C to E) Single-cycle viral replication kinetics. Vero cell monolayers were infected at MOI = 5 and incubated in the presence of 0.1% human serum. Cell monolayers were harvested at the time points indicated. (C) The quantity of viral genomes present was evaluated by qPCR for UL27. (D and E) Infectious virion production (titer) was evaluated by plaque formation on U2OS (D) or Vero (E) cells. These data represent results from three independent experiments. Two-way ANOVA followed by Tukey’s multiple-comparison test was applied. (F and G) Protein production. Vero cell monolayers were infected at MOI = 5 for 6 h (F) or 24 h (G). Whole-cell lysates were subjected to immunoblot analysis with the following antibodies: gC (UL44), gD (US6), gE (US8), gH (UL22), four virion glycoproteins; VP5 (UL19), capsid protein; ICP8 (UL29), viral single-strand DNA-binding protein; HSV, viral antibody against whole HSV-1; GAPDH, cellular glyceraldehyde-3 phosphate dehydrogenase as a loading control.

Enhanced viral cell-to-cell spread contributes to increased plaque size in culture. (A) The rates of viral spread in Vero cells were compared for representative neonatal isolates, including large-plaque formers (green) and small-plaque formers (black). Vero cell monolayers were infected at MOI = 0.001 in the presence of 0.1% human serum, which was replenished every 24 h. (B and C) Samples were harvested at each time point, and viral titers were evaluated by plaque formation on U2OS cells (B) or Vero cells (C). These data represent results from three independent experiments. Two-way ANOVA was performed followed by Tukey’s multiple-comparison test. *, P < 0.0001 at 72 h. (D) In parallel experiments, HSV-positive cells (green) were evaluated by immunofluorescence. Cell nuclei are counterstained with DAPI (blue). Scale bar = 200 μm. Images are representative of results from three independent experiments. Images of the entire 10-mm coverslips were then captured and stitched to create a composite image (see Fig. S2). (E) The total number of immunofluorescent (green) pixels was quantified for each coverslip. Two-way ANOVA was performed followed by Tukey’s multiple-comparison test. *, P < 0.05 at 72 h.

Neonatal HSV-2 genomes are genetically distinct from one another and encompass a broad range of known HSV-2 genetic diversity. A phylogenetic network constructed among neonatal HSV-2 genomes (A) or neonatal and adult HSV-2 genomes (B) reveals the wide genetic distribution of these unrelated isolates. HSV-2 genomes have been previously noted to lack geographic separation into clades (41, 42, 81). The network was created using SplitsTree4, from a MAFFT trimmed genome alignment. See Fig. S3 for a comparison tree constructed using a neighbor-joining (NJ) algorithm. See Table S1 for a complete list of accession numbers, geographic origins, and references for all 58 adult HSV-2 strains.

Neonatal HSV-2 proteins harbor consensus-level amino acid (AA) differences at a frequency similar to that seen between adult HSV-2 isolates. HSV-2 proteins are grouped by function, and the percentages of variable amino acids in each protein (representing the number of amino acid differences divided by protein length) are plotted for the 10 neonatal isolates (dark blue), for 10 representative adult HSV-2 isolates (light blue), and for all 58 adult HSV-2 genomes annotated in GenBank (clear outlines behind light blue bars). All adult HSV-2 genomes used for this comparison are listed in Table S1. See Table S2 for a numerical summary of nucleotide and AA differences observed in each set of neonatal or adult HSV-2 genomes.

Minor variants expand the range of neonatal HSV-2 coding diversity. (A) Scatter plot indicates the total number of minor variants (MV; y axis) observed in each neonatal isolate. MV are rare alleles that exist within each viral population, at a frequency that is <50% but above a 2% limit of detection (see Materials and Methods for details). The total number of MV on the left is separated into single-nucleotide polymorphism (SNP) versus insertion/deletion (indel) variants on the right (x axis). The genomic location of each SNP or indel variant is also summarized as follows: intergenic versus inside genes (genic) for indels and intergenic versus nonsynonymous or synonymous SNPs inside genes. (B) The frequency, or penetrance, of each minor variant was examined for each isolate. Data (x axis) were binned in increments of 5% (e.g., 2% to <5% frequency, 5% to <10% frequency, and so on) and are plotted according to the number of MV observed at each frequency (y axis). SNP and indel variants were combined for this analysis. (C) Stacked histograms show the number of genic MV (x axis) located in each HSV-2 coding sequence (gene; y axis). SNP and indel variants were combined for this analysis. UL3, UL11, UL35, and UL55 lacked any minor variants and are not included in the histogram. See Table S3 for a full list of SNP and indel MV position and frequency data.