JCM Accepts, published online ahead of print on 18 June 2008

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J. Clin. Microbiol. doi:10.1128/JCM.00536-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development and Validation of a Negative Strand-Specific RT-PCR Assay for Detection of a Chicken Strain of Hepatitis E Virus: Identification of Non-Liver Replication Sites

P. Billam, F. W. Pierson, W. Li, T. LeRoith, R. B. Duncan, and X. J. Meng^*

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342

^* To whom correspondence should be addressed. Email: xjmeng{at}vt.edu.

As a positive-strand RNA virus, hepatitis E virus (HEV) replication produces an intermediate negative-strand RNA. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study is to develop a negative strand-specific 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 used to standardize the assay. A nested set of primers were 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 were subsequently used to test thirteen convenient tissues collected sequentially at different days postinoculation from chickens experimentally-infected with avian HEV. In addition to liver, the negative strand-specific RT-PCR assay identified replicative viral RNA in gastraintinestinal tissues including colorectum, cecum, jejunum, ileum, duodenum and cecal tonsils. The detection of replicative viral RNA in these tissues indicates that HEV replicates in the gastraintinestinal tract, prior to reaching the liver, after oral ingestion of the virus. This is the first report on identification of extrahepatic sites of HEV replication in animals via natural route of infection. The assay should be of value for studying HEV replication and pathogenesis.

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