In silico and in vitro interrogation of a widely used HEV RT-qPCR assay for detection of the species Orthohepevirus A

Hepatitis E virus (HEV) infection is a public health concern worldwide, associated with waterborne outbreaks in developing countries and reported as an emerging zoonotic infection in high-income countries. A recent consensus proposal classified the isolates from human, swine, wild boar, deer, mongoose, rabbit and camel in seven genotypes within the species Orthohepevirus A. In this report a popular HEV RT-qPCR assay was assessed for the detection of the species Orthohepevirus A. In silico analysis of 189 complete genome sequences showed that the assay targets a highly conserved region in the Orthohepevirus A genome. Additionally, plasmid standards were constructed to test the effect of probe- and primer-binding site mutations in the assay performance. The assay proved robust enough to detect strains with mutations in the probe-binding site and in the 3′ end primer-binding site regions. A degenerate version of the reverse primer improves the performance of the assay particularly in the detection of HEV-5 and 6. The addition and detection of MS2 RNA in each RT-qPCR reaction monitored for amplification inhibition and did not affect the performance of the assay in the detection of the HEV RNA international standard. Therefore, the RT-qPCR assay can be confidently used for the RNA detection of the seven genotypes within the species Orthohepevirus A.