Identification of hepatitis E virus in clinical specimens: amplification of hydroxyapatite-purified virus RNA and restriction endonuclease analysis

Abstract

A multi-site nested reverse transcription and polymerase chain reaction (RT-PCR) followed by restriction endonuclease analysis (REA) was developed to identify hepatitis E virus (HEV) in clinical specimens. Four sets of primers were selected to amplify regions in the HEV genome supposed to encode the helicase, polymerase, and parts of the viral capsid protein. Digestion of the nested PCR products with HinfI, HaeII, AvaII, BglI, KpnI, SmaI, or EcoRI generated readily recognizable profiles that confirm the HEV sequences and/or distinguish the unique Mexico genotype (our positive control) from all other isolates (Asian genotype). In addition, the hydroxyapatite (HA) adsorption method was compared to other adsorption and extraction methods widely used to purify viral RNA from clinical specimens for RT-PCR. All methods presented the same sensitivity of recovery of HEV RNA, but only the adsorption methods efficiently removed fecal enzymatic inhibitors. The HA method gave the best results and was the most economic in terms of time, cost, manipulations and reagents. The method was validated by screening a small number of serum and fecal specimens available from patients with acute non-A,B,C hepatitis in Nepal. HEV RNA was identified in half (5/11) of the fecal specimens obtained from patients with evidence of recent HEV infection, but in none of the 14 patients without a serological marker for hepatitis E.