Construction of an infectious cDNA clone of a swine genotype 3 HEV Strain Isolated in Shanghai, China

Abstract

Objectives: Infectious cDNA clones are important tools for studying molecular mechanisms in RNA viruses. The aim of this study was to construct an infectious cDNA clone for SAAS-JDY5, which is a genotype 3 HEV strain of swine origin. Methods: Construction employed overlapping PCR and restriction analysis to ligate nine cDNA fragments into a full-length cDNA clone containing 14 mutations compared to the consensus HEV genome sequence. Megaprimer PCR-directed mutagenesis restored nine non-silent mutations back to the consensus sequence while the other five silent mutations were maintained as genetic markers. Results: HEV proteins were identified by an immunouorescence assay in Huh7 cells infected with capped RNA transcripts of the full-length cDNA clone, while HEV viremia, fecal HEV RNA and seroconversion were recorded in inoculated Sprague-Dawley rats. Conclusions: Our data confirmed the successful construction of an infectious cDNA clone of swine HEV strain pGEM4z-SAAS-JDY5, and support the use of rats as an HEV infectious model. © 2014 S. Karger AG, Basel.