A Novel Nanopore-Based Genotyping System for Norovirus GII: Validation and Application to Pediatric Gastroenteritis Cases in Moscow, Russia

Figure 1. Primer design strategy for norovirus GII whole-genome genotyping. (A) Genomic organization of target regions showing primer positions relative to reference strain NC_044045. Red arrows indicate the primary primer pair spanning the complete ~3.2 kb RdRp-VP1 junction (positions 3569–6749), while blue arrows show nested PCR primers for independent amplification of RdRp and VP1 regions. (B) Sequence logos of primer binding sites demonstrate nucleotide variability at degenerate positions (indicated by standard IUPAC codes), with reverse primers displayed as their target strand sequences, illustrating the conservation profile of primer annealing sites across 487 GII reference sequences indicating position-specific variability.

Figure 2. Schematic overview of the norovirus GII genotyping pipeline. The workflow processes Nanopore sequencing data through four key stages: (1) Data preprocessing. (2) Reference-based genotyping. (3) Analysis of the obtained data. (4) Phylogenetic tree construction.

Figure 3. Accuracy of minor genotype quantification. The scatter plot demonstrates the strong linear correlation (R2 = 0.99) between experimentally simulated and computationally predicted proportions of minor genotypes in mixed norovirus samples. Confidence intervals represent the margin of error observed across all tested concentrations (1–48%) being less than ±2%.

Figure 4. The pipeline’s genotyping accuracy against pairwise sequence divergence of different NoV-GII genotypes. The figure presents two symmetric heatmaps for (A) RdRP and (B) Capsid genotypes. The lower triangle of each matrix visualizes pairwise genetic distances (darker shades indicate higher sequence similarity). The upper triangle displays the results of in silico mixture experiments: for (A), two divergent RdRP sequences with a conserved capsid; for (B), two divergent capsid sequences with a conserved RdRP. Green cells indicate successful genotype discrimination by the pipeline, demonstrating high sensitivity, while light cells denote classification failure. The diagonal, representing zero divergence (self-comparison), provides a reference.

Figure 5. Temporal Distribution of Norovirus GII Genotypes. Stacked bar chart illustrating the annual prevalence of different Norovirus GII genotypes detected in pediatric patients with acute gastroenteritis in Moscow from 2021 to 2025 (n = 94). Each bar represents the proportional distribution of genotypes for a given year, summing to 100%. The specific genotype, defined by both capsid (e.g., GII.4) and polymerase (e.g., P16) regions, is indicated by the color key.

Figure 6. Phylogenetic analysis of norovirus sequences based on the RNA-dependent RNA polymerase (RdRp) (A) and capsid (B) regions. Major capsid genotypes are indicated by colored backgrounds and labels on the right, while the sampling year for each sequence is denoted by the font color. The consistent clustering of specific polymerase and capsid genotypes (e.g., GII.P16 with GII.4) supports strong genetic linkage, with noted exceptions indicating potential recombination events.