Hepatitis E Virus Genotype 3 in Quindío, Colombia: One Health Surveillance and First Genomic Characterization Reveal Cryptic Regional Divergence

Hepatitis E virus genotype 3 (HEV-3) is a zoonotic pathogen whose burden in Latin America remains underestimated. We conducted a cross-sectional One Health surveillance study (2019–2024) in Quindío, Colombia, testing 42 pig fecal, 8 farm runoff, 4 tributary water samples, and 5 clinical serum samples from patients with hepatitis of undetermined etiology by reverse transcription quantitative PCR (RT-qPCR). Positive and selected clinical/environmental samples underwent Oxford Nanopore MinION sequencing targeting the open reading frame 2 and 3 (ORF2/ORF3) junction, k-mer fingerprinting (k = 25), consensus generation, phylogenetic analysis, and Sanger confirmation. HEV RNA was detected in 8/42 pig fecal samples (19.0%), 4/8 runoff samples (50.0%), 1/4 tributary samples (25.0%), and 4/5 serum samples (80.0%). K-mer analysis identified strong HEV signals in two clinical samples (44.5% and 39.5% of reads) and wastewater (97.2%). Phylogenetic analysis classified Colombian isolates within HEV-3 (bootstrap 83%), while Sanger sequencing confirmed HEV-specific motifs. Two sequences were deposited in GenBank (PZ262436, PZ262437), representing the first Colombian HEV-3 genomic records. These findings demonstrate HEV-3 circulation across porcine, environmental, and human compartments and highlight substantial genomic divergence, supporting locally calibrated molecular approaches and strengthened One Health genomic surveillance in Colombia and Latin America.