Pathogen-focused metagenomic analysis reveals predominance of human rotavirus genotypes G3 and G12 in Zambian pediatric diarrhea cases.

Despite global improvements in water, sanitation, and rotavirus vaccination, rotavirus-associated diarrhea continues to cause significant morbidity and mortality among children in low-to-middle-income countries. Genomic surveillance is essential for evaluating vaccine efficacy and detecting emerging escape variants. In this study, we used VirCapSeq-VERT (VCS) to analyze rotavirus genetic diversity during Zambia's 2023 diarrhea surveillance. Stool samples from under five children with diarrhea were collected from health facilities across nine provinces. Out of 245 samples, 72 were rotavirus qPCR-positive with Ct <33 and underwent targeted viral enrichment and sequencing using VCS on the Illumina NextSeq2000. Bioinformatic analysis showed 70/72 strains had near complete genome constellations being genotyped as 45 Wa-like, 11 DS-like, and 14 reassortant strains. VP7 and VP4 analyses showed diverse genotypes (G1-G3, G8-G9, G12; P[4], P[6], P[8], P[11] clustering with vaccine and wild-type strains. Furthermore, G3 and G12 combined with P[4], P[6], and P[8] were the most predominant genotypes (35/70 and 13/70, respectively). Notably, nine samples had an M5 VP3 genotype with a 91% similarity to a simian rotavirus strain. Antigenic epitope analysis highlighted substitutions in P[6], G2, and G12, associated with immune escape. G3P[8] was the most common in severe cases. Fully vaccinated children showed significantly milder disease (p = 0.033). This study highlights VCS's utility in detecting viral diversity, reassortment, zoonotic transmission, and immune escape variants, providing crucial insights for assessing vaccine performance and public health strategies.