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Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 - ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n = 15), 3e (n = 4), and 3f (n = 15), representing the most prevalent subtypes in Europe. The mean [ORF3 - ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P = 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.