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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dc.contributor.authorBillam, P.en
dc.contributor.authorPierson, F. W.en
dc.contributor.authorLi, W.en
dc.contributor.authorLeRoith, Tanyaen
dc.contributor.authorDuncan, R. B.en
dc.contributor.authorMeng, Xiang-Jinen
dc.contributor.departmentVirginia-Maryland Regional College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology. Center for Molecular Medicine and Infectious Diseasesen
dc.date.accessed2015-11-28en
dc.date.accessioned2015-11-29T04:13:26Zen
dc.date.available2015-11-29T04:13:26Zen
dc.date.issued2008-06-18en
dc.description.abstractAs 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.en
dc.description.sponsorshipNational Institutes of Healthen
dc.description.sponsorshipAI074667en
dc.description.sponsorshipAI050611en
dc.format.mimetypeapplication/pdfen
dc.identifier.citationBillam, P. et al. (2008). 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. Journal of Clinical Microbiology, 46(8), 2630-2634. doi:10.1128/jcm.00536-08en
dc.identifier.doihttps://doi.org/10.1128/jcm.00536-08en
dc.identifier.issn0095-1137en
dc.identifier.urihttp://hdl.handle.net/10919/64220en
dc.identifier.urlhttp://jcm.asm.org/content/46/8/2630en
dc.language.isoenen
dc.publisherAmerican Society for Microbiologyen
dc.rightsIn Copyrighten
dc.rights.holderAmerican Society for Microbiologyen
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.titleDevelopment 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 sitesen
dc.title.serialJournal of Clinical Microbiologyen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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