Development and Validation of a Negative-Strand-Specific Reverse Transcription-PCR Assay for Detection of a Chicken Strain of Hepatitis E Virus: Identification of Nonliver Replication Sites

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Abstract
As a positive-strand RNA virus, hepatitis E virus (HEV) produces an intermediate negative-strand RNA when it replicates. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study was to develop a negative-strand-specific reverse transcription-PCR (RT-PCR) assay for the identification of extrahepatic sites of HEV replication. Briefly, a 494-bp fragment within the orf1 gene of a chicken strain of HEV (designated avian HEV) was amplified and cloned into a pSK plasmid. A synthetic negative-strand viral RNA was generated from the plasmid by in vitro transcription and was used to standardize the assay. A nested set of primers was designed to amplify a 232-bp fragment of the negative-strand viral RNA. The assay was found to detect up to 10 pg and 10(-5) pg of negative-strand HEV RNA in first- and second-round PCRs, respectively. The standardized negative-strand-specific RT-PCR assay was subsequently used to test 13 conveniently obtained tissue specimens collected sequentially on different days postinoculation from chickens experimentally infected with avian HEV. In addition to the liver, the negative-strand-specific RT-PCR assay identified replicative viral RNA in gastrointestinal tissues, including the colorectal, cecal, jejunal, ileal, duodenal, and cecal tonsil tissues. The detection of replicative viral RNA in these tissues indicates that after oral ingestion of the virus, HEV replicates in the gastrointestinal tract before it reaches the liver. This is the first report on the identification of extrahepatic sites of HEV replication in animals after experimental infection via the natural route. The assay should be of value for studying HEV replication and pathogenesis.

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