Nationwide Detection and Molecular Characterization of Hepatitis E Virus RNA in Retail Pork Meat in Japan

Hepatitis E virus (HEV) is a major zoonotic pathogen, with pigs serving as the principal animal reservoir. While consumption of raw or undercooked pig liver is a well-recognized risk factor, systematic data on HEV contamination in retail pork meat remain limited. In this study, 1546 retail pork samples collected from eight geographic regions across Japan were analyzed for HEV RNA using a validated nested RT-PCR method. Analytical sensitivity was determined using the World Health Organization (WHO) International Standard for HEV RNA, yielding a 95% limit of detection (LOD95) of 134 IU/g (95% CI: 105-193). Overall, 15 samples (1.0%) tested positive for HEV RNA, with no significant regional variation (0.4-1.8%; p = 0.8375) or difference between domestically produced (1.0%, 12/1260) and imported pork (1.0%, 3/286; p = 0.7478). Viral loads in quantifiable samples (n = 11) ranged from 9.3 × 10² to 1.0 × 10⁵ IU/g. Genotyping based on partial ORF2 sequences revealed subtype 3b as predominant (n = 7), followed by 3a (n = 5), 3f (n = 2), and 4c (n = 1). Each of the 15 strains showed high nucleotide sequence identity (98.8-100%) to its closest reported Japanese strain(s). Phylogenetic analysis showed that all strains clustered with their closest reported HEV strains, with high bootstrap support, despite genetic diversity within subtypes. These findings demonstrate that retail pork meat in Japan is contaminated with HEV RNA and may represent a potential source of human exposure. However, whether HEV RNA-positive retail pork meat contains infectious virus capable of causing human infection remains to be determined.