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
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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DOI：
10.1128/jcm.00536-08
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发表时间：
2008-08-01
影响因子：
9.4
通讯作者：
Meng, X. J.
中科院分区：
文献类型：
--
作者：
Billam, P.;Pierson, F. W.;Meng, X. J.
戊型肝炎病毒（HEV）是一种正链RNA病毒，在复制过程中产生一种中间负链RNA。因此，检测到负链病毒RNA指示HEV复制。本研究的目的是建立一种负链特异性逆转录聚合酶链反应（RT-PCR）检测戊型肝炎病毒复制的肝外位点。简言之，扩增HEV鸡株（命名为禽HEV）orf 1基因内的494 bp片段，并克隆到pSK质粒中。通过体外转录从质粒生成合成的负链病毒RNA，并用于标准化测定。设计了一套巢式引物，用于扩增负链病毒RNA的232 bp片段。在第一轮和第二轮PCR中，发现该检测方法分别检测到高达10 pg和10（-5）pg的负链HEV RNA。随后使用标准化的负链特异性RT-PCR检测13个方便获得的组织标本，依次收集在不同的日子接种后，从实验感染禽戊型肝炎病毒的鸡。除肝脏外，负链特异性RT-PCR检测还在胃肠道组织（包括结直肠、盲肠、空肠、回肠、十二指肠和盲肠扁桃体组织）中鉴定出复制型病毒RNA。在这些组织中检测到复制型病毒RNA表明，口服摄入病毒后，HEV在到达肝脏之前在胃肠道中复制。这是第一次报告的确定肝外网站的戊型肝炎病毒复制的动物实验感染后，通过自然途径。该方法对研究HEV的复制和致病机制具有一定的参考价值。
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.